Development of Food Chemistry Natural Products and Nutrition Research

Development of
Food Chemistry,
Natural Products,
and Nutrition
Research
Edited by
Antonello Santini and Nicola Cicero
Printed Edition of the Special Issue Published in Foods
www.mdpi.com/journal/foods
Development of Food Chemistry,
Natural Products, and
Nutrition Research
Development of Food Chemistry,
Natural Products, and
Nutrition Research
Special Issue Editors

Antonello Santini
Nicola Cicero
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin
Special Issue Editors
Antonello Santini Nicola Cicero
University of Napoli Federico II University of Messina
Italy Italy
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Contents
About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Antonello Santini and Nicola Cicero

Development of Food Chemistry, Natural Products, and Nutrition Research: Targeting
New Frontiers
Reprinted from: Foods 2020, 9, 482, doi:10.3390/foods9040482 . . . . . . . . . . . . . . . . . . . . . 1
Francesco Giuseppe Galluzzo, Gaetano Cammilleri, Alessandro Ulrici, Rosalba Calvini,

Andrea Pulvirenti, Giovanni Lo Cascio, Andrea Macaluso, Antonio Vella, Nicola Cicero,
Antonella Amato and Vincenzo Ferrantelli
Land Snails as a Valuable Source of Fatty Acids: A Multivariate Statistical Approach
Helena Maria Pinheiro-Sant’Ana, Pamella Cristine Anunciação, Clarice Silva e Souza,

Galdino Xavier de Paula Filho, Andrea Salvo, Giacomo Dugo and Daniele Giuffrida
Quali-Quantitative Profile of Native Carotenoids in Kumquat from Brazil by
HPLC-DAD-APCI/MS
Reprinted from: Foods 2019, 8, 166, doi:10.3390/foods8050166 . . . . . . . . . . . . . . . . . . . . 17
Gyung-Rim Yong, Yoseph Asmelash Gebru, Dae-Woon Kim, Da-Ham Kim, Hyun-Ah Han,
Young-Hoi Kim and Myung-Kon Kim
Chemical Composition and Antioxidant Activity of Steam-Distilled Essential Oil and
Glycosidically Bound Volatiles from Maclura Tricuspidata Fruit
Archimede Rotondo, Giovanna Loredana La Torre, Giacomo Dugo, Nicola Cicero,

Antonello Santini and Andrea Salvo
Oleic Acid Is not the Only Relevant Mono-Unsaturated Fatty Ester in Olive Oil
Ping-Chen Tu, Chih-Ju Chan, Yi-Chen Liu, Yueh-Hsiung Kuo, Ming-Kuem Lin and
Meng-Shiou Lee
Bioactivity-Guided Fractionation and NMR-Based Identification of the Immunomodulatory
Isoflavone from the Roots of Uraria crinita (L.) Desv. ex DC
Inga Matulyte, Aiste Jekabsone, Lina Jankauskaite, Paulina Zavistanaviciute,

Vytaute Sakiene, Elena Bartkiene, Modestas Ruzauskas, Dalia M. Kopustinskiene,
Antonello Santini and Jurga Bernatoniene
The Essential Oil and Hydrolats from Myristica fragrans Seeds with Magnesium
Aluminometasilicate as Excipient: Antioxidant, Antibacterial, and Anti-inflammatory Activity
Joanna Zielińska-Wasielica, Anna Olejnik, Katarzyna Kowalska, Mariola Olkowicz and

Radosław Dembczyński
Elderberry (Sambucus nigra L.) Fruit Extract Alleviates Oxidative Stress, Insulin Resistance, and
Inflammation in Hypertrophied 3T3-L1 Adipocytes and Activated RAW 264.7 Macrophages
v
Jin-Woo Jeong, Seon Yeong Ji, Hyesook Lee, Su Hyun Hong, Gi-Young Kim, Cheol Park,
Bae-Jin Lee, Eui Kyun Park, Jin Won Hyun, You-Jin Jeon and Yung Hyun Choi
Fermented Sea Tangle (Laminaria japonica Aresch) Suppresses RANKL-Induced
Osteoclastogenesis by Scavenging ROS in RAW 264.7 Cells
Agata Campisi, Rosaria Acquaviva, Giuseppina Raciti, Anna Duro, Milena Rizzo and
Natale Alfredo Santagati
Antioxidant Activities of Solanum nigrum L. Leaf Extracts Determined in In Vitro
Cellular Models
Gabriel López-Garcı́a, Antonio Cilla, Reyes Barberá, Amparo Alegrı́a and Marı́a C. Recio
Effect of a Milk-Based Fruit Beverage Enriched with Plant Sterols and/or
Galactooligosaccharides in a Murine Chronic Colitis Model
Wan-Sup Sim, Sun-Il Choi, Bong-Yeon Cho, Seung-Hyun Choi, Xionggao Han, Hyun-Duk
Cho, Seung-Hyung Kim, Boo-Yong Lee, Il-Jun Kang, Ju-Hyun Cho and Ok-Hwan Lee
Anti-Obesity Effect of Extract from Nelumbo Nucifera L., Morus Alba L., and Raphanus Sativus
Mixture in 3T3-L1 Adipocytes and C57BL/6J Obese Mice
Alexandra Galetović, Francisca Seura, Valeska Gallardo, Rocı́o Graves, Juan Cortés, Carolina
Valdivia, Javier Núñez, Claudia Tapia, Iván Neira, Sigrid Sanzana and Benito Gómez-Silva
Use of Phycobiliproteins from Atacama Cyanobacteria as Food Colorants in a Dairy
Beverage Prototype
Mailing Rivera, Alexandra Galetović, Romina Licuime and Benito Gómez-Silva

A Microethnographic and Ethnobotanical Approach to Llayta Consumption among Andes
Feeding Practices
vi
About the Special Issue Editors
Antonello Santini Ph.D., is Professor of Food Chemistry and Analysis of Food and Nutraceuticals and of
Food Chemistry at the Department of Pharmacy and at the Department of Agriculture at the University of
Napoli Federico II, Napoli, Italy, respectively. He is also Visiting Professor at the Albanian University of
Tirana, Albania. He holds a Ph.D. in Chemical Sciences. His research areas of interest are supported by many
international collaborations, mainly in the fields of food; food chemistry, nutraceuticals, and functional food;
safety; supplements; recovery of natural bioactive compounds using eco-sustainable and environmentally
friendly techniques from agro-food by products; nanocompounds; nanonutraceuticals; food risk assessment,
safety, and contaminants; mycotoxins and secondary metabolites; food analysis; and chemistry and food
education. He is responsible for funded research projects and for general cultural agreements established
between the University of Napoli Federico II and many universities worldwide, and external evaluator of
funded research projects for Italian and International Institutions. His research activity is documented by more
than 200 papers published in reputed peer-reviewed international journals. He is a member of the European
Food Safety Authority EFSA, ERWG, Parma, Italy; of the Italian Authority for Food Safety (CNSA), Italian
Ministry of Health, Rome Italy; of the Managing Board, Italian Chemistry Society (SCI) Division of Teaching
(DD-SCI), Rome, Italy; and Expert Member for Chemistry, EurSchool, European Commission, Bruxelles,
Belgium.
Nicola Cicero is Senior Researcher in Food Chemistry with the Department of Biomedical Sciences,
Dental and Morphological and Functional images, section S.A.S.T.A.S., at the University of Messina,
Italy. He holds two Ph.D. degrees: Enogastronomical Sciences and Tourism, Territory, and
Environment. He is teaching Oil and Wine in Mediterranean Food Habits and Fermentation
Biotechnology at the University of Messina, Italy. He is an expert in the quality and safety of agro-
food products. He has published about 100 publications, all in relevant peer-reviewed internationally
reputed journals. His research interests mainly focus on food, their quality assessment and safety
including the food and agro-food chain products, contaminants of organic and inorganic origin, and
analysis of food matrices. He has a relevant background of participation in international congresses
in the food area as chair or organizing and scientific committee member. He is an external evaluator
for the assessment of the scientific research activity of the Italian research system and has a wide
structured network of international collaboration in the food and environment area of interest. He is
a funder member of the scientific and technical committee of the spin-off company named
Science4Life srl and is an active consultant in the food industry.
vii
foods
Editorial
Development of Food Chemistry, Natural Products,
and Nutrition Research: Targeting New Frontiers
Antonello Santini 1, * and Nicola Cicero 2
1 Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy
2 Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Polo
Universitario Annunziata, 98125 Messina, Italy; ncicero@unime.it
* Correspondence: asantini@unina.it; Tel.: +39-81-253-9317
Received: 3 April 2020; Accepted: 4 April 2020; Published: 12 April 2020
Abstract: The Special Issue entitled: “Development of Food Chemistry, Natural Products, and
Nutrition Research” is focused on the recent development of food chemistry research, including
natural products’ sources and nutrition research, with the objectives of triggering interest towards new
perspectives related to foods and opening a novel horizon for research in the food area. The published
papers collected in this Special Issue are studies that refer to different aspects of food, ranging from
food chemistry and analytical aspects, to composition, natural products, and nutrition, all examined
from different perspectives and points of view. Overall, this Special Issue gives a current picture
of the main topics of interest in the research and proposes studies and analyses that may prompt
and address the efforts of research in the food area to find novel foods and novel applications and
stimulate an environmentally-friendly approach for the re-use of the by-products of the agro-food
area. This notwithstanding, the main challenge is currently addressed to achieve a full comprehension
of the mechanisms of action of food components, the nutrients, outlining their high potential impact
as preventive and/or therapeutic tools, not only as a source of macro- and/or micro-nutrients, which
are necessary for all the metabolic and body functions.
Keywords: foods; food analysis; nutrition; natural products; food supplements;

nanocompounds; nutraceuticals
The complete understanding of food matrices encompasses the analytical aspects and the
composition analysis, both of which are of paramount importance considering that emerging
new technologies and techniques in food analysis, chemometric techniques, and methods for food
authentication can allow obtaining a great amount of accurate and precise data. This potentially affects
the changes in consumer preferences and expectations, as well as the analysis of food innovations and
their impact on the global market [1,2].
Nonetheless, the frontier of the food chemistry has impacts with new challenges, which range
from: (i) novel foods; (ii) how adequate food safety may be determined; (iii) how nutritional intakes
evolve over time and are influenced by global dynamics; (iv) the novel delivery systems of the food
containing health beneficial compounds, which can have a great impact on health conditions, besides
their nutritional value and importance; (iv) natural sources and their waste or by-products’ recovery
and re-use [3,4].
The challenge to understand the mechanism of action of food micronutrients and of the secondary
metabolites involved in the chemistry of the food, especially when it is ingested, is currently triggering the
interest of researchers worldwide. The complete understanding of the metabolic pathway of foodstuffs,
which are complex matrices formed by many different substances, as well as the complete comprehension
and assessment of the effects that food has on the body’s metabolism are still open challenges.
Foods 2020, 9, 482; doi:10.3390/foods9040482 www.mdpi.com/journal/foods

Foods 2020, 9, 482
The analytical details and knowledge of all the minor food components and/or contaminants
of different origins at a very high resolution give important information, especially on food safety
and quality parameters. Nonetheless, the actual perspectives of the research in food chemistry reveal
an emerging interest in a new interdisciplinary approach that involves the contribution from different
disciplines both in the food and natural products areas. Natural products are and have been a primary
source in many cases, not only of nutrients, but also of remedies for millennia. An example is the growing
interest towards the re-use of by-products from industrial processing of food and foodstuff to recover
biologically-active substances to obtain derived products originating from food matrices and that may
be useful to support/supplement the diet. Nutraceuticals are an outstanding example of this emerging
trend in the food chemistry area. The use or re-use of food industry by-products, as well as the recovery
of biologically-active compounds are receiving growing attention in view of the great interest towards
the green economy and the optimization of the available resources. In this perspective, foodstuff and
agro-food industry by-products’ re-use can play a major role. A new challenging opportunity is to
explore and substantiate with detailed chemical composition data and clinical data the mechanisms and
modes of action of the active substances contained in food.
These aspects are relevant for maintaining well-being and preventing, by their use, the onset of
diseases due to poor diet/food habits [5–16]. Safety is also a major challenge, as well as obtaining the
complete or increased bioavailability of substances derived from food. From this perspective, the interest
towards nanomaterials is emerging. Due to their remarkable properties, these are currently considered
novel emerging tools to be used in the food area [17]. An interesting work has been reported recently
regarding nanomaterials’ application to foodstuff and outlining possible beneficial effects on health [18].
Nanopharmaceuticals can be considered as an illuminating example, which have led to a great
change in the pharmaceutical industry and have had a great impact also on nutraceuticals. There is
increasing growth in the study of nanocompounds including nutraceuticals derived from food matrices
as phytocomplexes to obtain improved delivery, bioavailability, and effects. As a consequence, many
recent research works are addressed towards the use of nanotechnologies applied to food-derived
nutraceuticals, building up the innovative area of new emerging products: nanonutraceuticals [19,20].
Nanotechnology could be used for the proficient delivery of bioactive substances contained in
food with the aim to improve their bioavailability, thereby increasing the possible health benefits.
The advantages of nanotechnology applied to nutraceuticals are efficient encapsulation, smart delivery
to the target, and release from a nanoformulation. For instance, research on the encapsulation of
nutraceuticals into biodegradable, environmentally-friendly nanocarriers is ongoing to increase their
absorption and therapeutic potential [21].
Nanonutraceuticals are a promising tool and a new frontier for the future research in the food area,
widening the horizon of foods to a new perspective, focusing on the active substances contained in food
matrices and on complete understanding of their mechanisms of action in the body. These food-derived
novel compounds should be assessed in order to maintain their properties at the nano level to target
better bioavailability and efficacy, naturally, notwithstanding the due attention to guarantee both
safety and efficacy. Follow-up studies, as well as clinical and nutritional studies to evaluate possible
unwanted effects would be necessary, and there is a long way to go in targeting the above-mentioned
points [22–27].
The papers that make up the Special Issue cover a wide range of topics. The application of an innovative
analytical technique based on Nuclear Magnetic Resonance (NMR) experiments called Multi-Assignment
Recovered Analysis (MARA)-NMR to extra-virgin olive oil allowed the quantitative assessment of the oil’s
chemical composition, opening a wide range of applications [28].
The study of the Fatty Acid (FA) profile of wild Theba pisana, Cornu aspersum, and Eobania
vermiculata land snail samples, examined by Gas Chromatography with a Flame Ionization Detector
(GC-FID), put into evidence a high content of Polyunsaturated Fatty Acids (PUFAs), indicating their
potential as functional food constituents [29].
Foods 2020, 9, 482
The study of the native carotenoid composition in kumquat (Fortunella margarita) from Brazil
determined for the first time by a HPLC-DAD-APCI/MS (High Performance Liquid Chromatography-Diode
Array Detector-Atmospheric Pressure Chemical Ionization/Mass Spectrometry) allowed identifying and
quantifying eleven carotenoids, some present in the free form and some in their esterified form [30].
The Special Issue includes studies addressing natural compounds and essential oils. In particular,
nutmeg (Myristica fragrans) has been studied with the aim of comparing the antioxidant, antimicrobial,
and anti-inflammatory activity of the hydrolats and essential oil obtained by hydrodistillation in the
presence and absence of magnesium aluminometasilicate as an excipient [31].
The essential oil obtained from Maclura tricuspidata fruit revealed the relevant antioxidant activities
of the steam-distilled essential oil and the glycosidically-bound aglycone fraction when studied with
the Gas Chromatography–Mass Spectrometry (GC–MS) technique [32].
Functional food ingredients were exploited in the study on Uraria crinita by screening its
metabolites using immunomodulatory fractions from the root methanolic extract in combination with
bioactivity-guided fractionation and NMR-based identification [33].
Other manuscripts published in the present Special Issue evaluated the capacity of Elderberry fruit
(EDB) extract to decrease the elevated production of reactive oxygen species in hypertrophied 3T3-L1
adipocytes, evidencing a crucial role in the development of obesity and accompanying metabolic
dysfunctions [34].
A study on the sea tangle (Laminaria japonica Aresch), a brown alga, used as a functional food
ingredient in the Asia-Pacific region, allowed assessing how fermented sea tangle extract was effective
on the receptor activator of the nuclear factor-κB (NF-κB) ligand using RAW 264.7 mouse macrophage
cells [35].
In addition, another interesting study contained in the Special Issue evaluated the antioxidant and
anti-adipogenic activities of another vegetal matrix, namely a mixture of Nelumbo nucifera L., Morus
alba L., and Raphanus sativus, with a complete updated in vitro and in vivo study [36].
The anti-inflammatory potential effect of plant sterols from enriched milk-based fruit beverages
(with or without galactooligosaccharides in an experimental mouse model of chronic ulcerative colitis)
was proposed, evidencing a great beneficial effect in mice against colitis [37].
Along the same lines, another interesting paper addressed foods in traditional medicine with
antioxidant potential, in particular the assessment of the antioxidant effect of leaf extracts of Solanum
nigrum L. [38].
The topics of the Special Issue expand the horizon of food research, also examining other
applications of vegetal matrices. An example is the paper dedicated to the study of Llayta, a biomass
of the colonies of Nostoc cyanobacterium grown in the wetlands of the Andean highlands, harvested,
sun-dried, and used as an ingredient for human consumption, which revealed great potential as
a functional food ingredient due to its relevant content of essential amino acids and polyunsaturated
fatty acids [39].
The collection of papers is completed with one study addressing also industrial food applications,
especially for industries interested in replacing artificial dyes with natural pigments; cyanobacterial
phycobiliproteins as water-soluble colored proteins to be used as natural eco-sustainable pigments
were shown to have great potential in this area of interest [40].
Author Contributions: All the authors contributed equally in the conceptualization, assessment, visualization,
and writing of the text.
Conflicts of Interest: No conflicts of interest, financial or otherwise, are declared by the authors.
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37. López-García, G.; Cilla, A.; Barberá, R.; Alegría, A.; Recio, M. Effect of a Milk-Based Fruit Beverage Enriched
with Plant Sterols and/or Galactooligosaccharides in a Murine Chronic Colitis Model. Foods 2019, 8, 114.
[CrossRef]
38. Campisi, A.; Acquaviva, R.; Raciti, G.; Duro, A.; Rizzo, M.; Santagati, N. Antioxidant Activities of Solanum
nigrum L. Leaf Extracts Determined in In Vitro Cellular Models. Foods 2019, 8, 63. [CrossRef]
39. Rivera, M.; Galetović, A.; Licuime, R.; Gómez-Silva, B. A Microethnographic and Ethnobotanical Approach
to Llayta Consumption among Andes Feeding Practices. Foods 2018, 7, 202. [CrossRef]
40. Galetović, A.; Seura, F.; Gallardo, V.; Graves, R.; Cortés, J.; Valdivia, C.; Núñez, J.; Tapia, C.; Neira, I.;
Sanzana, S.; et al. Use of Phycobiliproteins from Atacama Cyanobacteria as Food Colorants in a Dairy
Beverage Prototype. Foods 2020, 9, 244. [CrossRef]
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
foods
Article
Land Snails as a Valuable Source of Fatty Acids: A
Multivariate Statistical Approach
Francesco Giuseppe Galluzzo 1 , Gaetano Cammilleri 1,2, *, Alessandro Ulrici 2 , Rosalba Calvini 2 ,
Andrea Pulvirenti 2 , Giovanni Lo Cascio 1 , Andrea Macaluso 1,2 , Antonio Vella 1 , Nicola Cicero 3 ,
Antonella Amato 4 and Vincenzo Ferrantelli 1,2
1 Istituto Zooprofilattico Sperimentale della Sicilia, via Gino Marinuzzi 3, 90129 Palermo, Italy;
francescogiuseppe92@gmail.com (F.G.G.); giovanni.locascio71@gmail.com (G.L.C.);
andrea.macaluso@izssicilia.it (A.M.); laboratorio.residui@gmail.com (A.V.);
vincenzo.ferrantelli@izssicilia.it (V.F.)
2 Dipartimento di Scienze della Vita, Università degli studi di Modena e Reggio Emilia, Via Università 4,
41121 Modena, Italy; alessandro.ulrici@unimore.it (A.U.); rosalba.calvini@unimore.it (R.C.);
andrea.pulvirenti@unimore.it (A.P.)
3 Dipartimento SASTAS, Università degli studi di Messina, Polo Universitario dell’Annunziata,
98168 Messina, Italy; ncicero@unime.it
4 Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Università degli Studi di
Palermo, Viale delle Scienze, 90128 Palermo, Italy; antonella.amato@unipa.it
* Correspondence: gaetano.cammilleri86@gmail.com; Tel.: +39-328-8048262
Received: 4 November 2019; Accepted: 5 December 2019; Published: 12 December 2019
Abstract: The fatty acid (FA) profile of wild Theba pisana, Cornu aspersum, and Eobania vermiculata
land snail samples, collected in Sicily (Southern Italy), before and after heat treatment at +100 ◦ C
were examined by gas chromatography with a flame ionization detector (GC-FID). The results show
a higher content of polyunsaturated fatty acids (PUFAs) in all of the examined raw snails samples,
representing up to 48.10% of the total fatty acids contents, followed by monounsaturated fatty acids
(MUFAs). The thermal processing of the snail samples examined determined an overall reduction of
PUFA levels (8.13%, 7.75%, and 4.62% for T. pisana, C. aspersum and E. vermiculata samples, respectively)
and a species-specific variation of saturated fatty acid (SFA) contents. Oleic acid remained the most
abundant FA of all of the snails species examined, accounting for up to 29.95% of the total FA content.
A relevant decrease of Ñ3/Ñ6 ratio was found only for T. pisana samples. The principal component
analysis (PCA) showed a separation of the snail samples in terms of species and heat treatment.
The results of this work suggest land snails as a valuable source of MUFA and PUFA contents and
boiling as appropriate treatment, according to the maintenance of healthy properties.
Keywords: fatty acids; land snails; GC-FID; heat processing; principal component analysis
1. Introduction
Terrestrial gastropods, commonly named land snails, constitute a niche food product traditionally
appreciated by many European countries, especially France and Italy. The use of land snails as food
is still steadily growing, and 26,000 tons of snails were imported from Africa and countries in the
Middle East [1]. Cornu aspersum, Eobania vermiculata, and Theba pisana are the land snail species most
consumed in Italy [2]. Land snails are consumed in different ways all over the world, but the principal
cooking procedures recognized are roasting and boiling, according to the traditions of the countries.
According to Milinsk et al. [3], there is a correlation between land snails’ diet and their nutritional
values. Recently, increasing attention was paid to the fatty acid composition, due to nutritional and
health-related aspects [4–8]. However, few studies are available about the fatty acid (FA) profile in

Foods 2019, 8, 676
land snails [3,9,10] and, as far as we know, no data have been reported regarding the fatty acid profile
of T. pisana. Snails are commonly consumed in different ways after boiling due to the risk posed by the
possible presence of potentially pathogenic microorganisms [2]. The cooking temperature can influence
the nutritional aspect of mollusks [11]. At present, there are too few studies about the influence of heat
processing (such as boiling) on the nutritional composition of land snails.
In this context, the present work aimed at evaluating the fatty acids content of wild C. apsersum,
T. pisana, and E. vermiculata samples collected in Sicily (Southern Italy). Furthermore, the effect of
boiling on the fatty acid composition was evaluated to have a comprehensive nutritional evaluation of
this product after processing.
2. Materials and Methods
2.1. Reagents and Standards

All chemicals, solvents, and reagents employed were of analytical grade (≥99.9%). Acetone,
hexane, diatomaceous earth, sodium sulfide nonahydrate, methanol, and hydrochloric acid were
purchased from Sigma-Aldrich (Amsterdam, Holland). All of the gas used for gas chromatography
(GC) analysis was pure (≥99.9995%). Water used for the separation of fatty acid methyl esters (FAMEs)
phase was bidistilled in Milli-Q® Integral 5 (Merck KGaA, Darmstadt, Germany). FA standards were
purchased from Sigma-Aldrich (Amsterdam, Holland). The 10,000 mg/L standards were prepared by
diluting 100 mg of a pure standard solution with 10 ml of n-hexane. A mixture of FA standards was
used for the identification of each peak.
2.2. Sample Collection and Preparation

A total of 128 samples of C. aspersum, 400 samples of T. pisana, and 162 samples of E. vermiculata,
were collected from Palermo provinces (Sicily, Southern Italy) in 2018 during July for C. aspersum,
August for T. pisana, and September for E. vermiculata to have the maximum assimilation efficiency
according to the literature [12–14]. The shell of the snail samples was removed and only the meat was
considered for the chemical analysis. The meat of the snail samples was grouped into three pools
according to the species, then homogenized by a vertical mixer B-400 (Büchi, Flawil, Switzerland) and
stored at −10 ◦ C for 24 h to prevent a decrease in fatty acid content during the storage period [15].
The FA content of each sample pool was determined both raw and after cooking at 100 ◦ C with boiled
water for 30 s. The entire procedure of analysis is shown in Figure 1.
Figure 1. Scheme of the cooking process of the land snails samples collected.
2.3. Extraction of Fatty Acids and Gas Chromatography with a Flame Ionization Detector (GC-FID) Analysis
An amount of 10 ± 0.1 g of each pool of samples was placed in a glass of polypropylene and
mixed with diatomaceous earth (Sigma-Aldrich, Amsterdam, Holland). The mixture was transferred in
Foods 2019, 8, 676
an accelerated solvent extraction (ASE) ASE 200 cell (Thermo Fisher, Waltham, Massachusetts,
USA). The ASE operating conditions were set up as follows: 20 mL of hexane/acetone, 70:30;
extraction temperature 120 ◦ C for 6 min with a pressure of 120 pound per square (PSI).
The extract was filtered (size 240 nm) and dehydrated in rotavapor (Büchi, Flawil, Switzerland) at
+40 ◦ C. For the preparation of FAME, 100 mg of the oil extracted was trans-esterified in a pyrex tube by
using 2 mL of HCl/MeOH (2:98 v/v) to obtain the fatty acid methyl esters (FAMEs). The solution was
mixed in a vortex for 1 min and put in the oven at 120 ◦ C for 1 h. After cooling, 2 mL of bidistilled water
and 1 mL of hexane were added, and the mixture was centrifuged at 300 rpm for 1 min. Approximately
1 mL of the upper n-hexane phase was transferred in a vial and injected in gas chromatography (GC)
with a flame ionization detector (FID).
Each pool of samples was examined in triplicate by GC-FID analysis. The analysis was carried
out by a Trace GC/ULTRA HP 5890 GC + 7673 A/S (Thermo Fisher, Waltham, Massachusetts, USA);
a Famexax column (30m × 0.25 mm i.d. × 0.25 μm df) was used for the separation. A flame ionization
detector (FID) and ChromQuest 4.2.1 software (Thermo Fisher Scientific, Waltham, Massachusetts,
USA)were used for the qualification and quantification of the analytes. The injector port and the
detector temperatures were 220 ◦ C and 230 ◦ C, respectively. The split ratio was 1:20. The flow rates of
compressed air and hydrogen were 350 mL min−1 and 35 mL min–1 , respectively. The carrier gas was
helium (1.5 mL min−1 ). The oven temperature was programmed at a rate of 6.0 ◦ C min−1 from 130 to
225 ◦ C, held for 15 s.
Individual FAME was identified by comparison with the chromatographic behavior of authentic
standards by the formula:
TR = TR st ± 0.5 (1)
where TR is the determined retention time (min), and TR st is the retention time for each FA standard.
The relative percentages of the fatty acids were also determined. Quantitation of individual FAs is thus
based on the comparison of their peak areas (Ai), and the peak area of a suitable standard. The relative
percentages of fatty acids (C) were determined by the formula:
A
C = 100 (2)
A
2.4. Validation of the GC-FID Method

The repeatability mean and standard deviation of the analytical procedure were all calculated
according to Taverniers et al. [16]. Separated FA standards were used to calculate the mean retention
times (RTs) in the FID detector. The precision of the quantitative method was checked by the
repeatability test, based on ten series of experiments [17]. The area of each peak was measured and
corrected manually. The relative percentage of the fatty acids was also determined by comparing their
peak areas.
2.5. Data Collection and Statistical Analysis

The data were expressed as g/100g FA in fat extracted and grouped according to species and
treatment (raw T. pisana, C. aspersum, and E. vermiculata and cooked T. pisana, C. aspersum, and E.
vermiculata). The variation of fatty acids after heat treatment was calculated as follow:

Fab
Fa% = 100 − 100 (3)
Faraw
where Fa% is the variation (expressed as a percentage), Fab and Faraw are the fatty acid content in boiled
and raw samples, respectively (expressed as mg/100 g).
Before calculating the principal component analysis (PCA) model, the erucic acid variable was
removed from statistical analysis because its presence was found only in raw T. pisana samples. All of the
variables were pre-treated by Pareto scaling [18,19], in order to have a compromise between highlighting
Foods 2019, 8, 676
the contribution of the most abundant analytes and keeping at the same time the information brought
by the less abundant ones. The PCA model was calculated using the software PLS-Toolbox ver. 8.6
(Eigenvector Research Inc., Wenatchee, WA, USA), running in the MATLAB environment (ver. 9.3, The
Mathworks Inc., Natick, MA, USA).
3. Results
3.1. Fatty Acid Profiles

The fatty acid contents of the land snail samples examined are shown in Table 1. Seventeen FAs
were found in all of the species examined: Six saturated fatty acids (C14:0 , C16:0 , C17:0 , C18:0 , C20:0 , C22:0 ),
six monounsaturated (C14:1 , C16:1 , C17:1 , C20:1 , C18:1Ñ:9 ), and six polyunsaturated (C18:2Ñ:6 , C18:3Ñ:3 , C20:2 ,
C20:4 , C20:5 , C22:6 ). Erucic acid (C22:1 ) was found only in raw T. pisana samples.
Table 1. Fatty acids contents (mean ± SD; g/100 g FA) in fat extracted from the land snails species
examined (n = 3 replicates of each pool of samples). SFA = Saturated fatty acids, MUFA = monounsatured
fatty acids, PUFA = polyunsaturated fatty acids.
T. pisana T. pisana C. aspersum C. aspersum E. vermiculata E. vermiculata

Fatty Acid
Raw Boiled Raw Boiled Raw Boiled
Myristic (C14:0 ) 0.73 ± 0.01 1.06 ± 0.01 0.76 ± 0.08 0.59 ± 0.03 0.81 ± 0.01 0.63 ± 0.00
Palmitic (C16:0 ) 12.63 ± 0.04 15.75 ± 0.16 16.02 ± 0.27 13.2 4 ± 0.36 14.63 ± 0.13 13.31 ± 0.00
Margaric (C17:0 ) 1.02 ± 0.01 1.22 ± 0.07 1.13 ± 0.05 1.35 ± 0.07 1.36 ± 0.07 1.04 ± 0.03
Stearic (C18:0 ) 5.41 ± 0.08 6.31 ± 0.96 7.72 ± 0.2 7.24 ± 0.14 7.66 ± 0.03 7.59 ± 0.01
Arachidic (C20:0 ) 0.63 ± 0.00 0.69 ± 0.04 0.71 ± 0.03 0.39 ± 0.02 0.81 ± 0.02 0.37 ± 0.00
Behenic (C22:0 ) 0.31 ± 0.00 0.29 ± 0.01 0.64 ± 0.04 0.19 ± 0.00 0.31 ± 0.02 0.39 ± 0.00

SFA 20.72 25.32 26.97 23.01 25.58 23.35
Myristoleic (C14:1 ) 0.53 ± 0.00 0.59 ± 0.02 0.52 ± 0.03 0.65 ± 0.03 0.23 ± 0.01 0.58 ± 0.02
Palmitoleic (C16:1 ) 0.50 ± 0.02 0.27 ± 0.05 0.32 ± 0.08 1.35 ± 0.06 0.40 ± 0.04 0.37 ± 0.01
Eptadecenoic (C17:1 ) 0.52 ± 0.01 0.54 ± 0.04 0.81 ± 0.04 1.08 ± 0.04 1.01 ± 0.03 1.10 ± 0.00
Eicosenoic (C20:1 ) 0.37 ± 0.00 0.26 ± 0.03 0.37 ± 0.04 0.50 ± 0.02 0.17 ± 0.05 0.48 ± 0.00
Erucic (C22:1 ) 0.52 ± 0.00 - - - - -
Oleic (C18:1ω:9 ) 28.83 ± 0.08 28.83 ± 0.46 23.79 ± 1.72 29.95 ± 0.46 26.03 ± 0.63 29.71 ± 0.07

MUFA 31.27 30.49 25.81 33.53 27.86 32.24
Linoleic (C18:2 ω6 ) 18.78 ± 0.02 21.35 ± 0.10 22.15 ± 0.13 19.07 ± 0.16 21.94 ± 0.13 19.20 ± 0.02
Linolenic (C18:3 ω3 ) 7.64 ± 0.02 8.87 ± 0.16 15.78 ± 0.09 15.14 ± 0.20 12.40 ± 0.41 15.53 ± 0.00
Eicosadienoic (C20:2 ) 5.29 ± 0.02 5.44 ± 0.04 3.98 ± 0.02 4.64 ± 0.02 4.21 ± 0.40 4.69 ± 0.00
Arachidonic (C20:4 ) 6.28 ± 0.08 7.17 ± 0.19 4.26 ± 0.20 4.18 ± 0.01 6.40 ± 0.52 4.27 ± 0.03
Eicosapentaenoic (C20:5 ) 9.85 ± 0.01 1.15 ± 0.51 0.62 ± 0.07 0.40 ± 0.05 1.30 ± 0.06 0.43 ± 0.01
Docosahexaenoic (C22:6 ) 0.18 ± 0.01 0.21 ± 0.00 0.43 ± 0.01 0.13 ± 0.00 0.33 ± 0.03 0.29 ± 0.01

PUFA 48.10 44.19 47.22 43.56 46.56 44.41
ω3/ω6 0.58 0.30 0.55 0.56 0.43 0.58
PUFA/SFA 2.32 1.75 1.75 1.89 1.82 1.90
3.2. Fatty Acids of Raw Samples

The polyunsaturated fatty acid (PUFA) content of raw T. pisana, C. aspersum, and E. vermiculata was
48.10 g/100 g, 47.22 g/100 g, and 46.56 g/100 g, respectively, representing the most abundant class of fatty
acids, followed by monounsaturated fatty acid (MUFA) in T. pisana and E. vermiculata (31.27 g/100 g
and 27.86 g/100 g, respectively) and saturated fatty acid (SFA) in C. aspersum (26.97 g/100 g).
The main PUFA components were C18:2ω6 (18.78–22.15 g/100 g), C18:3ω3 (7.64–15.78 g/100 g),
and C20:2 (3.98–5.29 g/100 g). Linoleic acid (C18:2 ω6) represents the most abundant PUFA showing a
range between 7.64 and 15.78 g/100 g. A high level of eicosapentaenoic acid (C20:5) was determined in
T. pisana samples (9.85 g/100 g).
The MUFA profiles obtained for all the species examined consisted of C14:1 (0.23–0.53 g/100 g), C16:1
(0.32–0.5 g/100 g), C17:1 (0.52–1.01 g/100 g), C18:1ω:9 (23.79–28.83 g/100 g), and C20:1 (0.17–0.37 g/100 g).
Oleic acid was the main component of all the samples examined, representing 25% of the total fatty
acid content. Erucic acid was found only in raw T. pisana samples at low concentrations (0.52 g/100 g).
Foods 2019, 8, 676
Among the SFA, palmitic acid (C16:0) was the most abundant in all of the samples examined
(from 12.63 to 16.02 g/100 g), followed by stearic acid (5.41–7.66 g/100 g). The SFA profiles in all of
the species consisted of C14:0 (ranging from 0.73 to 0.81 g/100 g), C16:0 (12.63–16.02 g/100 g), C17:0
(1.02–1.36 g/100 g), C18:0 (5.41–7.72 g/100 g), C20:0 (0.63–0.81 g/100 g), and C22:0 (0.31–0.64 g/100 g).
The raw T. pisana samples showed the highest ω3/ω6 ratio (0.58), followed by E. vermiculata (0.55)
and C. aspersum (0.43).
3.3. Fatty Acids after Heat Treatment

After boiling at +100 ◦ C, the FA profile of all of the species examined verified a decrease of PUFA
content up to 8.2%. Differently from PUFA, a species-specific modification of MUFA and SFA contents
was found.
In particular, T. pisana samples showed an increase of SFA content by 22.20%; only C22:0 verified
a decrease after boiling (from 0.31 to 0.29 g/100 g).
The MUFA contents decreased by 2.49%. No erucic acid was found after boiling. The PUFA
content decreased from 48.10 to 44.19 g/100 g (8.13%), with a significant reduction of C20:5 content
(from 9.85 to 1.15 g/100 g).
Regarding C. aspersum, the SFA content decreased by 14.68%, showing a reduction of C20:0 from
0.71 to 0.39 g/100 g; only C17:0 showed an increase from 1.13 to 1.35 g/100 g.
The total MUFA content increased by 29%. Palmitoleic acid (C16:1) showed a significant increase
from 0.32 to 1.35 g/100 g, followed by C14:1, C17:1, and C30:1.
The E. vermiculata samples verified a reduction of SFA content after boiling (8.72%), especially
for arachidic acid (C20:0) (from 0.81 to 0.37 g/100 g), followed by C17:0 (1.36–1.04 g/100 g) and C14:0
(0.81–0.63 g/100 g). The behenic acid (C22:0) content increased by 25.81%. The MUFA content verified
an increase from 27.86 to 32.24 g/100 g. Palmitoleic acid (C16:1) was the only MUFA that decreased,
from 0.40 to 0.37 g/100 g. The heat treatment of the E. vermiculata samples determined a decrease
of PUFA components of 4.61%. Eicosapentaenoic acid (C22:6) decreased significantly from 1.30 to
0.34 g/100 g. The MUFA fatty acids more compromised after boiling were palmitoleic acid (C16:1) for
E. vermiculata and T. pisana samples and myristoleic acid (C14:1) for C. aspersum. Oleic acid (C18:1Ñ9)
remained the main component of all the land snails species examined even after boiling. Moreover, no
oleic acid content variation was found after boiling for T. pisana samples.
Among the PUFA group, eicosapentaenoic acid (C20:5) showed the highest decrease in T. pisana
and E. vermiculata samples after the heat treatment, whereas docosahexaenoic acid (C22:6) decreased
up to 70% in C. aspersum.
A decrease of the ω3/ω6 ratio was found only for the T. pisana samples, whereas the E. vermiculata
and C. aspersum samples increased the ω3/ω6 ratio up to 35%. Finally, a reduction of the PUFA/SFA
ratio was found only for T. pisana samples.
3.4. Multivariate Analysis

Given the high number of fatty acids examined as variables, principal component analysis (PCA)
was used to explore the dataset structure and to obtain more information on the variables that mainly
influence sample similarities and differences after heat treatment. The PCA model calculated after
Pareto scaling showed that the data group variation is visible in the first two principal components,
accounting for 97.82% of total data variance. Figure 2, which reports the PC1 vs. PC2 score plot
(Figure 2a), together with the corresponding loading plot, highlights that PC1 alone explains about
94% of data variance. This extremely high value, together with the fact that all the variables have
positive loading values along PC1 (Figure 2b), reflect the high positive correlation among the most
significant part of the considered variables, so PC1 describes the prevailing trend of the analyzed FAs
for the samples examined.
Foods 2019, 8, 676
Figure 2. PC1 vs. PC2 score (a) and loading (b) plots of FA contents of the land snail samples examined,
according to the species and treatment (raw vs. boiled).
The score plot shows differences related both to species and to heat treatment. PC1 describes a
decrease in the number of fatty acids for each species after boiling. The variation is more marked for
the T. pisana samples, which in general show the highest amount of unsaturated fatty acids (UFAs).
In fact, both raw and boiled T. pisana samples lie at positive values of PC1, whereas all E. vermiculata
and C. aspersum samples lie at negative values of PC1; this is due to the fact that, notwithstanding the
significant decrease of FA content, boiled T. pisana still has a UFA content higher than raw E. vermiculata
and raw C. aspersum. These last two samples have essentially the same overall amount of FAs and
boiling leads to a more marked decrease for C. aspersum than for E. vermiculata.
T. pisana shows an opposite trend compared to the two other species considering the position
along PC2 of the samples before and after boiling. Recalling that the percentage of variance explained
by PC2 (3.93%) is much lower than the percentage of variance explained by PC1 (93.89%), PC2 accounts
for the differences between the different species in the variations of the FA compositions after boiling,
beyond the overall decrease, explained by PC1.
These variations of the FA profile can be examined more in-depth by means of the corresponding
loading plot reported in Figure 2b. In this plot, the variables that are far from the origin contributed to
the systematic variability explained by the PCA model, whereas variables close to the origin (like, e.g.,
C17:1, C14:0, C20:2, C20:1, C22:6, etc.) did not show a systematic trend.
The significant variations after boiling for all of the three species examined are related to PUFA,
MUFA, oleic acid, SFA, and linoleic acid, which show higher PC1 values. PC2 showed a different
variation of the fatty acid contents between the T. pisana samples and the other two species. A more
considerable variation of eicosapentaenoic (C20:5) acid, MUFA, and oleic acid was found for T. pisana
samples, whereas E. vermiculata and C. aspersum showed a more significant variation of SFA, linoleic acid,
palmitic acid, and linolenic acid.
Foods 2019, 8, 676
4. Discussion
Fatty acids are ubiquitous molecules in biological systems. They play several roles in metabolism,
as structural components in membrane lipids, and as precursors of some molecules like prostaglandins
and eicosanoids [20]. The dietary intake in favor of PUFA and MUFA instead of SFA is correlated to a
significant minor risk of cardiovascular disease (CDV) and can lead to health benefits [21]. Fatty acids
are a minor nutritional parameter of snail meat [4,9,10]. However, all of the raw snail samples examined
in this work showed a low SFA content, in accordance to what was reported by Szkucik et al. [10] in
farmed C. aspersum samples from Poland, but in contrast to what was found in wild Helix pomatia
samples from Southern Turkey [9].
According to the literature, the factors of critical importance for the snail meat FA profile are
the snail genus and its collection site. Interspecies differences in fatty acid composition were also
confirmed in this work for the MUFA contents. In particular, the T. pisana raw meat samples showed
C20:5 contents up to 16 times higher than C. aspersum and E. vermiculata samples; these differences
could be due to the different ecological aspects of the species examined. It is well known that T. pisana
is an agricultural pest in many parts of the world [21,22], feeding on a wide range of agricultural
plants, including cereals with high UFA contents [14,23]. Differently from T. pisana, C. aspersum
and E. vermiculata appear to be selective polyphagous organisms, preferring plants of the Poaceae
family [12,24,25]. Other studies have confirmed how the feeding regimen would affect the fatty acid
composition [4], verifying significant variations of MUFA contents related to the increase of soybean
oil as feed supply in reared C. aspersum samples. Feeds that include corn, sunflower, or soybean rich in
ω6 acids were shown to increase the content of these FAs in meat.
Nevertheless, the wild snail samples examined in this work showed high contents of UFA,
constituting up to 79% of the total fatty acids. The level of PUFA in edible snails was found to be higher
than SFA and MUFA, according to what was found in Helix lucorum and Limax flavus [26]. Our results
are contrary to what was reported by Ekin et al. [27] in free-living Melanopsis praemorsa snails of
Anatolia (Turkey) that showed a lower level of UFA and higher SFA contents. Essential fatty acids such
as linoleic acid, α linolenic acid, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) were
determined. These fatty acids show protection effects against cardiovascular disease [28,29]; however,
the current intakes of EPA and DHA in European populations appear to be below the recommended
daily allowance (RDA) [29].
The thermal processing of the snail samples analyzed determined an overall reduction of PUFA
levels and a species-specific variation of MUFA and SFA contents, in contrast to what was found by
Szkucik et al. [10] in farmed C. aspersum samples from Poland, verifying a significant increase of the SFA
levels. The PUFA amounts of the samples analyzed decreased up to 7.98%, with a significant decrease
of the C 20:5 contents in T. pisana samples. Nevertheless, PUFA remained the principal component,
accounting for 44% of the total fatty acid contents in all of the species examined.
Among the MUFA, oleic acid (C18:1) remained the most abundant fatty acid of all of the snails
species examined, even after heat processing.
Regarding the SFA contents, our results appear to comply with what was reported by
Purwaningsih et al. [11] in mollusk muscles, showing that the SFA composition depends primarily on
the snail species, rather than the way of cooking. The heat treatment of T. pisana samples determined a
decrease of Ñ3/Ñ6 ratio from 0.58 to 0.3, reaching a value lower than the minimal ratio recommended
by the WHO [30]. However, the heat treatment allowed obtaining a total degradation of toxic fatty
acid as the erucic acid. Animal tests showed that the ingestion of oils containing erucic acid could lead
to a heart disease called myocardial lipidosis. Other potential effects observed in animals (changes in
liver, kidney, and skeletal muscle weight) occur at slightly higher doses.
Contrary to what was found by Szkucik et al. [10], the C. aspersum samples examined in this work
showed a considerable decrease of the relative amounts of SFA after heat treatment, favoring an increase
of the relative amounts of MUFA. The E. vermiculata samples showed a similar behavior of C. aspersum
samples after heat treatment, showing a decrease of 8.72% for SFA and 4.66% for PUFA, and an increase
Foods 2019, 8, 676
of the amounts of MUFA. A reduced PUFA content could be caused by the autoxidation mechanisms
initiated by temperature rise in meat during it is cooking [10,31]. Furthermore, these modifications
appear to be related to the process temperature, the cooking time, and the internal temperature reached
by the meat [31–34].
Principal component analysis allowed to depict the significant sources of variability of the dataset
analyzed using two Principal Components (PCs), which showed a clear separation of the land snail
samples according to species and heat treatment. The high percentage of variance explained by PC1
(93.89%) reflects the fact that the investigated variables were highly correlated, showing a general
decrease due to heat treatment; this variation was much more pronounced for T. pisana than for the
species C. aspersum and E. vermiculata. The highest variation after heat treatment common to all the
three species was related to PUFA, MUFA, oleic acid, and linoleic acid.
5. Conclusions
To the best of our knowledge, this work reports, for the first time, the fatty acid composition
of T. pisana samples and their variation as a result of heat processing, and the first time of fatty acid
composition in E. vermiculata, T. pisana, and C. aspersum collected in Sicily (Southern Italy). The results
showed a species-specific variation of FA contents in the land snails samples examined after boiling,
showing the highest UFA decrease in T. pisana samples.
The results demonstrate that the land snail species examined could be a good source of MUFA and
PUFA and their contents are species-specific. Boiling could be an adequate cooking procedure for land
snail consumption according to retained nutritional and healthy criteria (PUFA contents and ω-6/ω-3
ratio). Furthermore, the boiling process can safeguard consumers against potentially pathogenic
microorganisms. Given that boiling losses seem to be related to cooking time and temperature,
further studies are needed to find the best cooking condition in order to preserve the best nutritional
composition criteria of land snails.
Author Contributions: Conceptualization, F.G.G. and G.C.; methodology, F.G.G., A.M. and G.C.; validation, A.M.,
A.A., N.C. and G.L.C.; formal analysis F.G.G. and G.C.; investigation, A.P., A.U. and N.C.; data curation, A.U., R.C.,
G.C. and F.G.G.; writing—original draft preparation, F.G.G. and G.C.; writing—review and editing N.C., A.P., A.A.,
V.F. and A.V.; visualization, V.F. and A.A.; supervision, V.F.; project administration, V.F.; funding acquisition, V.F.
Funding: This research was funded by MINISTERO DELLA SALUTE, grant number RC IZS SI 16/15.
Acknowledgments: The authors thank Barbara Randisi for the technical support.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to
publish the results.
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foods
Article
Quali-Quantitative Profile of Native Carotenoids in
Kumquat from Brazil by HPLC-DAD-APCI/MS
Helena Maria Pinheiro-Sant’Ana 1 , Pamella Cristine Anunciação 1 , Clarice Silva e Souza 1 ,
Galdino Xavier de Paula Filho 2 , Andrea Salvo 3, *, Giacomo Dugo 3 and Daniele Giuffrida 3
1 Departamento de Nutrição e Saúde, Universidade Federal de Viçosa, Avenida P.H. Rolfs, s/n,
Viçosa 36571-000, Brazil; helena.santana@ufv.br (H.M.P.-S.); nutripamella@gmail.com (P.C.A.);
cla_souzabio@yahoo.com.br (C.S.e.S.)
2 Departamento de Educação, Universidade Federal do Amapá, Rodovia Juscelino Kubitschek, Km 02,
Jardim Marco Zero, Macapá 68903-419, Brazil; galdinoxpf@gmail.com
3 Department, of Biomedical and Dental Sciences and Morphofunctional Imaging, University of
Messina (Italy), V.le Annunziata, 98168 Messina, Italy; dugog@unime.it (G.D.); dgiuffrida@unime.it (D.G.)
* Correspondence: asalvo@unime.it; Tel.: +39-090-676-6880
Received: 3 April 2019; Accepted: 14 May 2019; Published: 16 May 2019
Abstract: In this study the native carotenoids composition in kumquat (Fortunella margarita) (peel +
pulp) from Brazil was determined for the first time by a HPLC-DAD-APCI/MS (high performance liquid
chromatography-diode array detector-atmospheric pressure chemical ionization/mass spectrometry),
methodology. Eleven carotenoids were successfully identified and quantified in kumquat: four
carotenoids in the free form and seven carotenoids in the esterified form. β-citraurin-laurate
was the carotenoid found in the highest content (607.33 μg/100 g fresh matter), followed by
β-cryptoxanthin-laurate (552.59 μg/100 g). The different esterified forms of β-citraurin and
β-cryptoxanthin represented 84.34% of the carotenoids found, which demonstrates the importance
of esterification in natural fruits. β-carotene and free xanthophylls (β-cryptoxanthin, lutein and
zeaxanthin) represented 5.50% and 14.96%, respectively, of total carotenoids in kumquat. The total
carotenoid content of kumquat from Brazil was very high (2185.16 μg/100 g), suggesting that this fruit
could contribute significantly to the intake of important bioactive compounds by the population.
Keywords: Fortunella margarita; citrus; carotenes; xanthophylls; β-citraurin-laurate; β-cryptoxanthin-

laurate
1. Introduction
The citrus family is one of the first crops in the world, it is estimated that half of the marketed
production comes from the Americas and 12% comes from the Mediterranean basin. Citrus cultivation
is thought to date back at least 4000 years and is mainly from the Asiatic south-east territories.
The estimated global citrus traffic for 2017–2018 was around 6 million tons. The most representative
cultures were Citrus sinensis (61%), Citrus reticulata (22%), Citrus limon (11%) and Citrus paradisi (6%).
In the Americas, the primacy of citrus production lies with Brazil followed by the United States. Sweet
oranges are grown in Brazil, mainly in the state of São Paulo, over an area of about 584000 hectares,
but also in the Amazonas area of northern Brazil, over about 2.7 hectares. [1].
The Citrus japonica, known by the common names of kincan (from Japanese kinkan) or cunquate
(from Chinese kumquat), is a small citrus fruit of the Rutaceae family [2]. It has four major cultivated
types, including Fortunella japonica, Fortunella margarita, Fortunella crassifolia, and Fortunella hindsii [3].
In eastern countries, this fruit is a part of the regular food habits of the population [4], but in Brazil
it is considered exotic, in addition to being little known and commercialized. Among the Brazilian
states, São Paulo has the largest production and commercialization of this fruit [5].

Foods 2019, 8, 166
Kumquats are native to Central China. They are oval or round fruits with peel and orange smooth.
Its flavor varies from acid to sweet. The fruit is rich in vitamins, carotene, pectin, calcium, phosphorus,
iron and flavonoids [6]. Kumquats are consumed preferably in natura, whole and in shell. It is also
used to make jellies, mousse, jams, marmalades, liqueurs and cachaça [7], preparation of syrups, sauces
and also, accompaniment in fruit salads and for landscaping purposes and ornamentation [8,9].
Citrus japonica has been used as a traditional folk medicine in Asian countries to reduce alcohol
intoxication and as antidepressants, so they are used either as medicines or as edible fruit [10]. Many
studies on antioxidant, antimicrobial and antitumor effects have been carried out on kumquats,
however identification of the bioactive compounds in the fruit has received little attention [11].
The most elucidated chemical components in kumquat described in the literature are phenolics
compounds and flavonoids. Different phenolic compounds and flavonoids are described in Fortunella sp.
by HPLC-MS [12,13]. These studies have shown a higher concentration of phenolics in fruit peels, with
luteolin and kaempferol being the main flavonoids found in Fortunella sp. [8,14–17].
Studies on carotenoids in kumquat are extremely limited [9,18–20]. Agócs et al. [18] studied the
qualitative and quantitative composition of carotenoids of kumquat and other citrus species. However,
the sample preparation of the carotenoids involved a saponification step, a procedure that does not
allow us to evaluate the native composition of the carotenoids.
Studies on the composition of carotenoids in foods are very important because they participate in
various biological processes in plants, such as photosynthesis, photomorphogenesis, photoprotection,
and development [21]. In animals, provitamin A carotenoids play an essential role in the synthesis
of retinol (vitamin A) [22], whereas the xanthophylls lutein and zeaxanthin have been associated in
humans with the prevention of age-ralated eye degenerations [23,24].
Carotenoids are molecules made up of a long chain of usually forty carbon atoms; they can by
divided into two classes: (a) non oxygenated one named carotenes [25] and (b) oxygenated one named
xanthophylls [26]. Moreover, the xanthophylls are usually esterified with fatty acids in nature.
The studies on the content of carotenoids in kumquats concern plants grown in Asia but data
are not available for fruits harvested in Brazil [18,19]. Therefore, this work aimed to determine the
complete qualitative and quantitative profile of the kumquat carotenoid native composition for fruits
collected in the rural area of Viçosa, Minas Gerais, Brazil, through liquid chromatography coupled to
the mass detector (HPLC-DAD-APCI-MS).
2.1. Chemicals
The standards of β-carotene, β-cryptoxanthin, lutein, zeaxanthin and physalein, Standard purity
was above 98% were purchased from Extrasynthese (Genay, France), and the solvents MeOH (Methanol),
MTBE (Methyl-t-butyl ether) and H2 O (Water) from Sigma-Aldrich (Milan, Italy).
2.2. Collection and Preparation of the Samples

The fruits of kumquat (Fortunella margarita) (Figure 1) were collected in the morning, in May
2017, in the rural area of Viçosa (latitude 20◦ 44’ 05” S and longitude 42◦ 51’ 27” W), Minas Gerais,
Brazil. Samples were collected in four repetitions of approximately 1 kg each. The fruit maturation
was determined according to Donadio et al. [27] and defined by the red-orange peel color and the
characteristic smell. In addition, ripe fruits were considered as those obtained after their natural fall of
the trees or fall after being lightly touched by the hands.
The species was identified with the help of taxonomists from the Universidade Federal de Viçosa
Herbarium through the Angiosperm Phylogeny Group IV [28], where it has already been cataloged
and registered in the Virtual Herbarium network with the following records: EAC 48987, HUCO 5197,
HPL 8977 and SP 42766.
Foods 2019, 8, 166
The samples were transported from the harvest site to the laboratory protected in styrofoam
boxes with blocks of ice, within two hours after collection. In the laboratory, the samples were selected
for appearance, excluding those with any epidermis injury or mechanical damage due to transport.
The fruits were removed from the seeds (peel + pulp) were homogenized in a food processor (RI 7625,
Philips, São Paulo Brazil), lyophilized (Liotop-LP510, Liobras, São Carlos, Brazil) and stored in plastic
containers with screw caps, covered with aluminum foil stored at −18 ± 1 ◦ C until further analyses.
Figure 1. Kumquat (whole fruit and cross-section) from Viçosa, Minas Gerais, Brazil.
2.3. Moisture Analysis

Moisture was determined in triplicate in the oven (SP 200, SP Labor® , São Paulo, Brazil), at 65 ± 1 ◦ C,
for approximately 72 h [29].
2.4. Extraction of Carotenoids

The carotenoid pigments were extracted from the lyophilized material (peel + pulp), according
to the recommended procedures by Rodrigues-Amaya et al. [30]. Three grams of the edible portion
of the samples (peel + pulp) were crushed by the use of a mortar and pestle, and a few drops of
distilled water were added and extracted to color exhaustion with 20 mL of acetone (7 times) in an
ultrasonic bath (Labsonic LBS 1-H22.5, Treviglio, Bergamo, Italy) for 10 min each time. Then, the extracts
were individually centrifuged (Awel MF20-R, Multifunction Refrigerated Centrifuge, Blain, France) at
4000 rpm, 5 ◦ C, for 10 min in order to withdraw clear solution on the top. The acetonic extracts were
pooled together was concentrated to about 25 mL, in a rotary evaporator (Buchi-heating bath B-491,
Buchi, Milan, Italy) at temperature below 35 ◦ C. The dry product was diluted with equal volumes
(25 mL) of a mixture of ethyl ether and hexane (1:1) and distilled water (50 mL) and worked up with a
separating funnel. The lipofilic phase, cleared by the hydrophilic impurities, was evaporated to dryness
using a rotary evaporator (Buchi-heating bath B-491 at 35 ◦ C, and the residue was dissolved in 2 mL of
MeOH/MTBE (1:1) and filtered in filter units (PTFE, 0.45μm, 13mm, Sigma Aldrich, Milan, Italy) prior
to HPLC analysis. Samples were stored at −20 ◦ C until they were analyzed.
2.5. Analysis of Carotenoids by HPLC-DAD-APCI-MS

The analysis was performed on an HPLC system (Shimadzu, Kyoto, Japan) equipped with a
CBM-20A controller, two LC-20AD pumps, a DGU-20A3R deaerator, a SIL-20AC autosampler, a CTO
Foods 2019, 8, 166
20AC column oven and an SPD-M20A photo diode array detector. The data were processed with the
Labsolution software. For MS analysis a mass spectrometer detector (LCMS-8040) was used, equipped
with an APCI (atmospheric pressure chemical ionization) interface, both in positive and negative
ionization mode.
The column used was YMC C30 (250 mm × 4.6 mm × 5 μm); the mobile phases: MeOH/MTBE/H2O
(81:15:4, solvent A), MeOH/MTBE/H2O (6:90:4, solvent B); the linear gradient used was: 0–100% B
from 0 to 140 min. The column temperature was maintained at 30 ◦ C. The flow was 0.8 mL/min and
the injection volume was 20 μL.
The UV-Vis spectra were acquired in the range 220–700 nm, while the chromatograms were
extracted at 450 nm (sampling frequency: 4.16 Hz, time constant: 0.64 s). The MS was set up as follows:
Scan, both APCI positive (+) and negative (-); atomized gas flow (N2): 2.0 L/min; drying gas flow:
5 L/min; Time of the event: 0.06 s; range m/z: 300–1200; interface temperature: 350 ◦ C; Desolvation line
(DL) temperature: 300 ◦ C; thermal block: 300 ◦ C. The samples were analyzed in triplicate.
2.6. Identification and Quantification of Carotenoids

Carotenoids were identified by their UV-Vis spectra, MS spectra, elution order, comparison with
the available standard and literature data.
The kumquat carotenoids quantification was performed from the analytical curves. External
standards quantitative determination of each compound was performed using all reference materials
listed in Section 2.1, in the concentration range from 5 to 50 μg/mL at six concentration levels. The
results were obtained from the average of three determinations and the CV% was below 8% in all the
LC measurements. The R coefficient for the calibration curves was always above 0.9962, with LOD
and LOQ values of 0.07 and 0.22 ppm for β-carotene, 0.1 and 0.33 ppm for β-cryptoxanthin, 0.06 and
0.18 ppm for lutein, 0.08 and 0.3 ppm for zeaxanthin, and 0.12 and 0.24 ppm for physalein, respectively.
3. Results and Discussion
3.1. Carotenoids Qualitative Profile of Brazilian Kumquat

Figure 2 shows the chromatographic profile of the carotenoid composition in not saponified
kumquat fruits extracts. The identified compounds are shown in Table 1, together with the UV–Vis
and MS spectra information. In Figure 3 are reported the UV-Vis (PDA) and mass spectrum of
β-citraurin-laurate and β-citraurin-myristate, detected in the kumquat carotenoid extracts. It can be
appreciated that the esterification does not affect the PDA spectra of β-citraurin.
Figure 2. Chromatographic profile of native carotenoids of kumquat from Brazil: peel + pulp.
Identification of the compounds are in Table 1.
Foods 2019, 8, 166
Table 1. Compounds identification for Figure 2 (kumquat from Brazil: peel + pulp).
Compound Identification Rt (min) PDA (λnm) MS (APCI-) m/z

1 Lutein 18.5 445, 473 568
2 Zeaxanthin 20.2 449, 476 568
3 β-cryptoxanthin 28.4 428, 451, 478 552
4 β-citraurin-caproate 29.9 454 586
5 β-citraurin-laurate 32.8 455 614
6 β-citraurin-myristate 36.2 453 642
7 β-carotene 38.1 426, 451, 476 536
8 β-citraurin-palmitate 40.4 455 642
9 β-cryptoxanthin-laurate 44.5 428, 450, 478 734
10 β-cryptoxanthin-myristate 47.3 428, 450, 477 762
11 β-cryptoxanthin-palmitate 50.9 428, 451, 478 790
Rt: retention time; PDA: photodiode array; λnm: wavelength of maximum absorption; MS: mass spectrometry;
APCI: atmospheric pressure chemical ionization.
Figure 3. UV-Vis (PDA) and mass spectrum (APCI negative) of β-citraurin-laurate (A) and β-citraurin-
myristate (B) of kumquat from Brazil.
Moreover, both the molecular ions [M]−• at respectively m/z 614 and m/z 642 relative to the
β-citraurin-laurate and β-citraurin-myristate esters, obtained in the negative APCI mode, are also
clearly shown in the same figure. Eleven different carotenoids were identified in kumquat from Brazil;
four different β-citraurin esters and three β-cryptoxanthin esters were identified.
The predominant carotenoids were β-citraurin-laurate and β-cryptoxanthin-laurate, both esterified
with lauric acid. The chemical structures of these carotenoids are shown in Figure 4. Interestingly, no
free β-citraurin was detected in the present study.
Foods 2019, 8, 166
Figure 4. Chemical structures of β-cryptoxanthin-laurate and β-citraurin-laurate, main carotenoids

detected in kumquat.
Shirra et al. [9] detected only 4 carotenoids in kumquat from Italy without saponification
(β-carotene, β-cryptoxanthin, lutein and zeaxanthin), which were also detected in the present study.
In contrast to our study, Agos et al. [18] reported only β-citraurin and β-cryptoxanthin in the free form
in kumquat from Hungary. However, these researchers used saponification after the extraction process
and did not quantify the carotenoid esters. Saponification may result in destruction or structural
transformation of carotenoids [31]. Huyskens et al. [19] studied the qualitative composition of kumquat
carotenoids from Israel by thin-layer chromatography and reported several carotenoids, including
β-citraurin, β-cryptoxanthin, lutein, β-carotene and zeaxanthin which were, also determined in the
present study. In addition, Huyskens et al. [19] reported that violaxanthin was the predominant
component in kumquat from Israel, whereas in our study β-citraurin-laurate was the major component.
Esterification with saturated fatty acids improves the stability of xanthophylls such as β-citraurin
and β-cryptoxanthin against heat and UV light, but does not affect their antioxidant activity [32].
During the storage and processing of the fruits the xanthophyll esters were more stable than the free
xanthophyll [33]. The pigment β-citraurin is responsible for the citrus reddish color, it derives from
of β-cryptoxanthin or zeaxanthin and accumulates in some citrus varieties [34,35]. In these fruits,
the accumulation of β-citraurin is not a common event, it is observed only in the flavedos of some
varieties during fruit ripening [34]. Frutita, a tropical fruit from Panama, showed a very high content
of β-citraurin [36].
Recent studies have shown that β-cryptoxanthin and β-citraurin esterified with lauric acid,
myristic acid and palmitic acid are found in the Mandarin Satsuma, [35].
The dietary intake of β-cryptoxanthin has been shown to prevent and reduce some pathologies
such as: cancer, diabetes and rheumatism due to its antioxidant activity [37–39]. Breithaupt et al. [40]
have verified that in chili, papaya, peach and persimmon, β-cryptoxanthin is mainly esterified with
saturated fatty acids. Furthermore, the bioavailability of β-cryptoxanthin esters is comparable to
the non-esterified form, since fatty acids can be effectively hydrolyzed by β-cryptoxanthin esters
before intestinal absorption in the human body [40]. In several citrus varieties, the esterified form
β-citraurin is found [35]. As already observed in another study [41], we have found β-cryptoxanthin
and β-citraurin in both free and esterified forms.
3.2. Carotenoids Quantitative Profile of Brazilian Kumquat

In the present study, β-citraurin-laurate was the carotenoid found in the highest content in
kumquat from Brazil (607.33 μg/100 g fresh matter, representing 27.80% of total carotenoids), followed
by β-cryptoxanthin-laurate (552.59 μg/100 g fresh matter, representing 25.31% of total carotenoids).
Thus, β-citraurin-laurate and β-cryptoxanthin-laurate represented 53.11% of the total carotenoids
Foods 2019, 8, 166
found. The different forms of β-citraurin (4 components esterified with fatty acids) and β-cryptoxanthin
(1 component in free form and 3 in esterified form) represented 84.34% of the carotenoids found in
kumquat from Brazil. Forms of β-citraurin and β-cryptoxanthin esterified with fatty acids accounted
for 79.54% of the total carotenoids in kumquat. These results show the importance of the study of the
intact carotenoids composition in different matrices (Table 2).
Table 2. Carotenoid content in kumquat from Brazil (peel + pulp).
Content in Lyophilized Content in Fresh Carotenoid

Compound Carotenoid
Kumquat (μg/100 g) * Kumquat (μg/100 g) * Composition (%)
1 Lutein 873.05 ± 93.51 144.35 ± 15.45 6.61
2 Zeaxanthin 469.55 ± 25.52 77.63 ± 4.21 3.55
3 β-Cryptoxanthin 634.93 ± 55.91 104.98 ± 9.25 4.80
4 β-Citraurin-caproate 721.81 ± 20.04 119.34 ± 3.32 5.46
5 β-Citraurin-laurate 3673.28 ± 81.78 607.33 ± 13.63 27.80
6 β-Citraurin-myristate 421.88 ± 9.52 69.75 ± 1.58 3.20
7 β-Carotene 726.96 ± 110.60 120.19 ± 18.29 5.50
8 β-Citraurin-palmitate 801.27 ± 66.35 132.48 ± 10.98 6.06
9 β-Cryptoxanthin-laurate 3342.25 ± 126.10 552.59 ± 20.75 25.31
10 β-Cryptoxanthin-myristate 927.77 ± 82.92 153.39 ± 13.71 7.02
11 β-Cryptoxanthin-palmitate 623.73 ± 46.34 103.13 ± 7.67 4.72
Total carotenes 726.96 120.19 5.50
Total free xanthophylls 1977.53 326.96 14.96
Total esterified xanthophylls 10511.99 1738.01 79.54
Total carotenoids 13,216.48 2185.16 100
* Mean of 3 repetitions ± standard deviation (SD). The carotenoid content in the fresh kumquat was calculated based
on the average moisture content (n = 3) of the lyophilized fruit (14.08%) and the fresh fruit (81.16%).
β-carotene was found in smaller amounts (120.19 μg/100 g fresh matter), representing 5.50% of total
carotenoids in kumquat, as well as free xanthophylls (β-cryptoxanthin, lutein and zeaxanthin), which
represented 14.96% of total carotenoids (326.96 μg/100 g) (Table 2). β-carotene and β-cryptoxanthin
possess provitamin A activity, playing a key role in human health [42]. Differently from our results,
Wang et al. [20] reported that β-cryptoxanthin was the major carotenoid present in kumquat cultivated
in Taiwan, while Schirra et al. [9] found lutein to be the major carotenoid present in kumquat from Italy.
Schirra et al. [9] found a lower concentration, in fresh matter, of β-carotene (33 μg/100 g),
β-cryptoxanthin (26 μg/100 g), lutein (44 μg/100 g) and zeaxanthin (24 μg/100 g) in kumquat from
Italy when compared to the concentrations of these carotenoids in kumquat from Brazil, reported
here for the first time. Wang et al. [41] found contents in dry matter that were much lower than
reported here, for lutein (9.9 μg/100 g), zeaxanthin (10.4 μg/100 g), β-cryptoxanthin (183 μg/100 g) and
β-carotene (131 μg/100 g) in kumquat cultivated in Taiwan. Carotenoid composition and contents in
fruits can be influenced by genetic factors, geographical regions, fruit processing, storage methods
Giuffrida et al. [25] and environmental conditions Lu et al. [26], which can explain the differences
found in these studies. Besides the climatic factors, the irrigation, soil conditions, fertilizers and
herbicides may also affect the carotenoids accumulation [26]. Regarding the carotenoids content in
food, Britton [43] have proposed the following classification ranges: low: 0–0.1 mg/100 g; moderate:
0.1–0.5 mg/100 g; high: 0.5-2 mg/100 g; very high: >2 mg/100 g. Thus, the total carotenoid content of
kumquat from Brazil was very high (2185.16 μg/100 g).
4. Conclusions
In this study the native carotenoids composition in kumquat (Fortunella margarita) from Brazil was
determined for the first time. Eleven native carotenoids in kumquat from the rural area of Minas Gerais,
Brazil were successfully identified and quantified by HPLC-DAD-APCI-MS. Four carotenoids in the
free form (β-carotene, β-cryptoxanthin, lutein and zeaxanthin) and 7 carotenoids in the esterified form
were identified (β-citraurin-caproate, β-citraurin-laurate, β-citraurin-myristate, β-citraurin-palmitate,
β-cryptoxanthin-laurate, β-cryptoxanthin-myristate, β-cryptoxanthin-palmitate). β-citraurin-laurate
and β-cryptoxanthin-laurate were the most abundant native carotenoids in kumquat from Brazil. The
Foods 2019, 8, 166
total carotenoid content of kumquat from Brazil was high (2185.16 μg/100 g), suggesting that this fruit
can contribute significantly to the ingestion of important bioactive compounds.
Author Contributions: Conceptualization, H.M.P.-S., P.C.A.; Methodology, C.S.e.S., G.X.d.P.F.; Writing-review

and editing, A.S.; Supervision, G.D., and D.G.
Funding: This research received no external funding.
Acknowledgments: The authors thanks the Fundação de Amparo à Pesquisa do Estado de Minas Gerais
(FAPEMIG), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de
Aperfeiçoamento de Ensino Superior (CAPES) and the Università degli Studi de Messina for financial support for
conduction of the study.
Conflicts of Interest: The authors declare no conflict of interest.
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foods
Article
Chemical Composition and Antioxidant Activity of
Steam-Distilled Essential Oil and Glycosidically
Bound Volatiles from Maclura Tricuspidata Fruit
Gyung-Rim Yong, Yoseph Asmelash Gebru, Dae-Woon Kim, Da-Ham Kim, Hyun-Ah Han,
Young-Hoi Kim and Myung-Kon Kim *
Department of Food Science and Technology, Chonbuk National University, Jeonju 54896, Jeonbuk, Korea;
rudfla1226@naver.com (G.-R.Y.); holden623@naver.com (Y.A.G.); eodns3344@gmail.com (D.-W.K.);
dadaham@naver.com (D.-H.K.); hha208@Korea.kr (H.-A.H.); yhoi1307@hanmail.net (Y.-H.K.)
* Correspondence: kmyuko@jbnu.ac.kr; Tel.: +82-63-270-25512
Received: 12 November 2019; Accepted: 5 December 2019; Published: 9 December 2019
Abstract: Essential oil obtained from Maclura triscuspidata fruit has been reported to have functional
properties. This study aimed at determining chemical compositions and antioxidant activities of
steam-distilled essential oil (SDEO) and glycosidically bound aglycone fraction (GBAF) isolated from
fully ripe M. triscuspidata fruit. SDEO was isolated by simultaneous steam distillation and extraction
(SDE). GBAF was prepared by Amberlite XAD-2 adsorption of methanol extract, followed by methanol
elution and enzymatic hydrolysis. Both fractions were analyzed by gas chromatography–mass
spectrometry (GC–MS). A total of 76 constituents were identified from both oils. Apart from fatty acids
and their esters, the SDEO contained p-cresol in the highest concentration (383.5 ± 17.7), followed by
δ-cadinene (147.7 ± 7.7), β-caryophyllene (145.7 ± 10.5), β-ionone (141.0 ± 4.5), n-nonanal (140.3 ± 20.5),
theaspirane A (121.3 ± 4.5) and theaspirane B (99.67 ± 9.05 μg/g). Thirteen carotenoid-derived
compounds identified in the SDEO are being isolated from M. triscuspidata fruit for the first time.
Out of the 22 components identified in GBAF, 14 were present only in the glycosidically bound
volatiles. Antioxidant activity of the GBAF was higher than that of SDEO. These results suggest that
glycosidically bound volatiles of M. triscuspidata fruit have a good potential as natural antioxidants.
Keywords: Maclura triscuspidata fruit; essential oil; glycosidically bound volatiles; gas chromatography-mass
spectroscopy (GC–MS); chemical composition; antioxidant activity
1. Introduction
Plant-derived essential oils are complex mixtures of volatile and semi-volatile organic compounds
characterized by diverse odors and chemical compositions depending on their origins. They are
traditionally obtained from various plant tissues including fruits, seed, leaves, flowers, roots, woods
and barks by means of hydrodistillation, steam distillation, solvent extraction or cold pressing [1,2].
Due to their organoleptic and biological properties, essential oils have been used as flavoring agents
and natural preservatives in foods since ancient times [3]. More recently, essential oils and some of
their isolated components are increasingly being used in various commercial products such as foods,
cosmetics, perfumes, household cleaning products and hygiene products, and medicinal applications [2].
These compounds have been reported to have various biological activities including antimicrobial,
antioxidant, antiviral, antiplatelet, antithrombotic, antiallergic, anti-inflammatory, antimutagenic, and
anticarcinogenic properties [4–6].
Lipid oxidation causes serious problems in foods by producing unpleasant flavors, discoloration,
decreasing nutritional quality and safety of foods through due to production of secondary oxidation
products that have harmful effects on human health [7]. The use of essential oils as natural antioxidants

Foods 2019, 8, 659
is a field of growing interest because of the fact that synthetic antioxidants such as butylated
hydroxyanisole (BHA) and butylated hydroxyltoluene (BHT) have been suspected of causing liver
damage and carcinogenesis when used at high levels in laboratory animals [8–11]. For this reason,
their use in the food industry has recently declined owing to safety concerns and consumer demand
for natural products.
Maclura tricuspidata (Carr.) Bur. (formerly known as Cudrania tricuspidata) which belongs to the
Moraceae family is a thorny tree native to East Asia including China, Japan and Korea. The leaves, root,
stem and fruit of this plant have been used in traditional herbal medicines to treat jaundice, hepatitis,
neuritis and inflammation in Korea [12]. Several beneficial effects of M. tricuspidata extracts have been
reported including anticancer [13,14], anti-inflammatory [15], antioxidant [16,17], and antidiabetes
effects [18]. Various bioactive compounds such as prenylated xanthones, phenolic acids and flavonoids
have already been identified from its leaves, root, stem and fruit [19–21].
The ripe fruits of Maclura tricuspidata which have a bright red color are edible with a floral
aroma and sweet taste. They have traditionally been used to prepare fresh juice, jam, wine, vinegar
and fermented alcoholic beverages in Korea. Previous studies have reported that the extracts and
components of M. tricuspidata fruits have strong antioxidant and free radical-scavenging activities in
an in vitro system [22,23]. The antioxidant activity of M. tricuspidata fruit extract is associated with
the presence of phenolic compounds such as flavonoids and phenolic acids [17,24]. We have recently
identified 18 polyphenolic compounds among which five parishin derivatives (gastrodin, parishin
A, B, C, E) identified for the first time in the fruit and confirmed their anti-oxidant potentials [25].
Essential oil obtained from the fruit by microwave-assisted hydrodistillation has also been reported
to have antioxidant activity through 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide, hydroxy
and superoxide radical scavenging activities [26]. Recently, Bajpai and colleaques [26] identified 29
compounds as major constituents in the essential oil isolated from M. tricuspidata fruit. Although the
chemical compositions and their antioxidant activities of essential oils from the stem and root of M.
tricuspidata were elucidated [26,27], the information on the chemical composition and antioxidant
activity of the essential oil of M. tricuspidata fruit is still very poor. Furthermore, it is known that
some volatile compounds in plants are present either in a free form and glycosidically bound forms
to sugar moiety [28,29]. In some plants, glycosidically bound volatiles have shown a more potent
antioxidant activity than essential oils [30,31]. Nevertheless, little is known about chemical constituents
and their antioxidant potentials of glycosidically bound aglycones in M. tricuspidata fruit. Therefore,
the objective of this study was to elucidate the chemical composition of steam-distilled essential oils
(SDEO), aglycone fraction and major compounds of aglycone fraction liberated from glycosidically
bound volatiles (GBAF) in M. tricuspidata fruit and their antioxidant potentials.
2.1. Reagents
n-Decanol, n-decyl-β-d-glucopyranoside, Amberlite XAD-2 polymeric resin (20–60 mesh), butylated
hydroxyanisole (BHA), butylated hydroxy toluene (BHT), ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl
(DPPH), 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tri(2-pyridyl)-
s-triazine (TPTZ) and saturated n-alkanes mixture (C7 –C30 ), were purchased from Sigma-Aldrich
Corp. (St. Louis, MO, USA). Authentic volatile chemicals were purchased from commercial sources
(Sigma-Aldrich and Wako Pure Chemical Industries, Ltd., Osaka, Japan). The other reagents used were
of analytical grade and were purchased from commercial sources.
2.2. Plant Materials

M. tricuspidata fruits were collected in late October 2017 at a fully mature stage from plants
cultivated in a farm located in Milyang district, Gyeongsangnam-do, Republic of Korea. A voucher
specimen has been deposited at the Herbarium of Department of Food Science and Technology, College
Foods 2019, 8, 659
of Agricultural Life Science, Chonbuk National University. The fruit was freeze-dried for 4 day. The
samples were powdered and stored in a freezer (−20 ◦ C) until use.
2.3. Isolation of Steam-Distilled Essential Oil

A powdered sample (100 g) and distilled water (2 L) were placed in a 3 L round flask. The essential
oil was isolated by means of simultaneous steam distillation and extraction at atmospheric pressure in
a modified Likens–Nickerson type apparatus using n-pentane-diethyl ether (1:1) containing n-decanol
(950 μg) as an internal standard for 2 h [32]. After the isolated oil was dried over anhydrous sodium
sulfate for 12 h, the solvent was concentrated to a volume of 0.5 mL using a Vigreaux column at 40 ◦ C
and thereafter was evaporated off under a stream of nitrogen. The resulting residue was redissolved
in 1 mL of n-pentane-diethyl ether (1:1) and subjected to gas chromatography (GC) and GC–mass
spectrometry (GC–MS) analysis.
2.4. Isolation of Free Volatiles and Glycosidically Bound Volatiles

The powdered sample (100 g) was homogenized with 300 mL of methanol for 1 min in a Waring
blender. The homogenate was centrifuged at 4500× g for 20 min. The residue was homogenized
with 300 mL of methanol followed by centrifugation as above. The supernatant was combined and
the solvent was concentrated to remove methanol under reduced pressure at 40 ◦ C. The residue was
dissolved in 100 mL of distilled water and was passed through a previously preactivated (with methanol)
Amberlite XAD-2 (20–60 mesh) adsorbent column (5 × 35 cm) at a flow rate of 3 mL/min according
to a previously reported method [33]. After the column was washed with 1.5 L of distilled water,
free volatiles (FV) and glycosidically bound volatile (GBV) fraction was isolated by sequentially eluting
with each 1 L of n-pentane:diethyl ether (1:1) and methanol, respectively. The FV fraction was dried
over anhydrous sodium sulfate for 12 h and filtered through filter paper. The filtrate was concentrated
to remove solvent under reduced pressure at 40 ◦ C. The resulting residue was redissolved in 1 mL of
n-pentane-diethyl ether (1:1). The methanol eluate designated as GBV was concentrated under reduced
pressure to dryness at 40 ◦ C. After residue was redissolved in 50 mL of 0.1 M citrate-phosphate buffer
(pH 4.8), the aqueous layer was washed triplicate with each 50 mL of n-pentane:diethyl ether (1:1)
to remove remaining free volatiles and added n-decyl-β-d-glucopyranoside (1900 μg) as an internal
standard. The GBF was hydrolyzed by Aspergillus niger cellulase (80 mg, 24 U as β-glucosidase) at
37 ◦ C for 36 h with gentle shaking. The liberated aglycones were isolated by liquid-liquid extraction
using ethyl acetate (50 mL × 3). After the liberated glycosidically bound aglycone fractioin (GBAF)
was dried over anhydrous sodium sulfate for 12 h, the solvent was evaporated using rotary evaporator
at 40 ◦ C. The resulting residue was dissolved in ethyl acetate. The extracts prepared were stored at
−20 ◦ C until use.
2.5. Gas Chromatography (GC) and GC–Mass Spectrometry (GC–MS) Analysis

GC analysis was performed on a Hewlett-Packard model 6890 series gas chromatograph, with a
flame ionization detector (FID), a split ratio of 1:30 using Agilent J&W DB-5MS fused silica capillary
column (30 m × 0.32 mm, i.d., 0.25 μm film thickness, Santa Clara, CA, USA) and Agilent J&W
Supelcowax 10 fused silica capillary column (30 m × 0.32 mm, i.d., 0.25 μm film thickness). The column
temperatures were programmed from 50 ◦ C to 230 ◦ C at 2 ◦ C/min and then kept constant at 230 ◦ C
for 20 min. The injector and detector temperatures were 250 ◦ C, respectively. The carrier gas was
nitrogen, at a flow rate of 1.0 mL/min. Peak areas were measured by electronic integration and the
concentrations of volatile compounds were expressed as n-decanol equivalent (assuming response
factor of all analytes was 1.0). The concentrations are to be considered only relative values as recovery
after extraction and calibration factors related to the standard were not determined [34,35].
The GC–MS analysis was performed on an Agilent Technologies 7890A GC and 5975C mass
selective detector operating in the EI mode at 70 eV, fitted with a DB-5MS fused silica capillary
column (30 m × 0.25, i.d., 0.25 μm film thickness) and Supelcowax 10 fused silica capillary column
Foods 2019, 8, 659
(30 m × 0.32 mm, i.d., 0.25 μm film thickness), respectively. Both column temperatures were
programmed from 50 ◦ C to 230 ◦ C at 2 ◦ C per minute and then kept constant at 230 ◦ C for 20 min.
The injector and ion source temperatures were 250 ◦ C. The carrier gas was helium at a flow rate of
1.0 mL/min. Identification of the compounds was achieved by comparing their retention times with
those of authentic standards and mass spectral data in Wiley7n,1 database (Hewlett-Packard, Palo Alto,
CA, USA), and NIST (National Institute of Standards and Technology, USDA) Webbook, and reported
retention indices in the literatures [36]. Retention indices of each compound was calculated by a
homologous series of saturated n-alkanes (C7 –C30 ) (concentration of 1000 μg/mL in n-hexane) under
the same conditions [37]. All compounds identified based on comparisons of only mass spectral data
were listed as tentatively identified.
2.6. Determination of Total Phenolic Content

Total phenol content of the sample was measured according to the method described by Chandra
et al. [38] with some modifications. Briefly, 20 μL of each fraction (at concentration of 1000 μg/1 mL
methanol) was mixed with 50% Folin–Ciocalteu phenol reagent (20 μL) in 96-well plates. After 5 min,
1 N sodium carbonate solution (20 μL) was added to the mixture and distilled water was added to
adjust the final volume to 200 μL. After incubation at room temperature (RT) in the dark for 30 min,
the absorbance of test sample against a blank was measured at 725 nm using a VersaMax enzyme-linked
immunosorbent assay (ELISA) microplate reader (Molecular Devices, LLC, San Jose, CA, USA). Total
phenol content was calculated based on a calibration curve of gallic acid. The results were expressed
as mg gallic acid equivalent (mg GAE)/g.
2.7. Antioxidant Activity
2.7.1. Preparation of Sample

The solvent in the test samples (SDEO, FV, GBV and GBAF) were removed under a nitrogen
stream. The resulting residues were dissolved in n-pentane:diethyl ether (1:1). BHA, BHT and ascorbic
acid all diluted to a concentration of 1000 μg per mL in methanol were used as positive controls for the
antioxidant activity assays.
2.7.2. DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Free Radical-Scavenging Activity

DPPH radical scavenging activity was determined according to the method described by Thaipong
et al. [39] with some modifications. For calculation of effective concentration EC50 value, a stock
solution of DPPH was freshly prepared by dissolving 240 mg DPPH in methanol (1000 mL) and the
working solution was prepared by diluting stock solution with methanol to obtain an absorbance of
1.1 ± 0.02 units at 517 nm using an ultraviolet–visible (UV–vis) spectrophotometer (Shimadzu UV-1601,
Osaka, Japan). 100 μL of the samples (SDEO, FV and TBAF) and chemicals were allowed to react with
0.1M Tris-HCl buffer (900 μL) and 500 μM DPPH solution (1000 μL) for 20 min at RT in the dark. Then
absorbance was taken at 517 nm using UV–vis spectrophotometer. The EC50 (μg/mL) were calculated
from the regression curves using six different concentrations (10–100 μg/mL) of samples and chemicals.
The results were expressed as EC50 value (μg/mL). As a blank, the test was repeated using buffer
instead of samples, and the DPPH radical-scavenging activity of the extracts was calculated against a
blank as follows:
DPPH radical-scavenging activity (%) = (1 − A0 /A1 ) × 100
where A0 and A1 are absorbance values of the test sample and control, respectively.
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2.7.3. ABTS (2,2 -Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid)) Free Radical-Scavenging Activity
ABTS free radical scavenging activity was determined by the methods of Thaipong et al. [39] with
some modifications. Briefly, a mixture of ABTS (7.4 mM) solution and potassium persulfate (2.6 mM)
solution in 1:1 ratio was kept at room temperature for 12 h under dark condition to form ABTS cation.
The solution was diluted by adding methanol to obtain an absorbance of 1.1 ± 0.02 at 734 nm. All the
required solutions were freshly prepared for each assay. 100 μL of the samples and chemicals were
added to 1400 μL of the diluted ABTS solution and the mixture was incubated at room temperature for
2 h in a dark. After the reaction, its absorbance was measured at wavelength of 734 nm. The results
were expressed as RC50 value (μg/mL), and also ABTS radical scavenging activity (%) was calculated
with the following equation:
ABTS radical scavenging activity (%) = (1 − A0 /A1 ) × 100
where A0 and A1 are absorbance values of the test sample and control, respectively.
2.7.4. Ferric-Reducing Antioxidant Power (FRAP)

Ferric-reducing power was determined using FRAP assay [40] with some modification. The FRAP
reagent was prepared by mixing 10 volume of 300 mM acetate buffer (pH 3.6) with 1 volume of 10 mM
TPTZ solution in 40 mM HCl and 1 volume of 20 mM ferric chloride solution. Sample extract (75 μL)
was added to 1425 μL of FRAP reagent. The reaction mixture was then incubated at RT for 30 min in a
dark. The reducing power was expressed as absorbance at 593 nm and RC50 values (μg/mL) of FRAP
were calculated from the regression lines using six different concentrations (10–100 μg/mL) in triplicate.
2.8. Statistical Analysis

All experiments were conducted in triplicate unless otherwise indicated and the results were
expressed as mean ± standard deviation (SD). The statistical analysis was conducted with SPSS (ver.
10.1) for Windows and a one-way analysis of variance (ANOVA). Duncan’s multiple range tests were
carried out to test any significant differences among various fruit maturity stages. Values with p < 0.05
were considered as significantly different
3.1. Chemical Composition of the Steam-Distilled Essential Oil (SDEO) Fraction

The yields of total SDEO and GBAF from M. tricuspidata fruit were 0.03 ± 0.01% and 0.37 ± 0.03%,
respectively. Table 1 shows the volatile compounds identified in the SDEO and GBAF isolated from M.
tricuspidata fruit along with their amounts and retention indices on DB-5MS (non-polar) and DB-WAX
(polar) column. A total of 55 compounds including 17 tentatively identified compounds were identified
in SDEO. The compounds that were found by only DB-5MS column but not by DB-WAX column
were considered as tentatively identified. The compounds were 4 alcohols, 14 aldehyde and ketones,
7 terpenoids, 13 carotenoid-derived compounds, 6 aromatic and phenolic compounds, 11 acids and 3
miscellaneous. With the exception of aliphatic acids and their esters such as palmitic acid, linoleic acid,
ethyl palmitate and linoleic acid, compounds with the highest concentration in the SDEO were p-cresol
(393.50 ± 17.70), followed by δ-cadinene (147.67 ± 7.50), β-caryophyllene (145.67 ± 10.50), β-ionone
(141.00 ± 4.40) and n-nonanal (140.33 ± 20.50 μg/g). In particular, 10 kinds of carotenoid-derived
compounds were identified in the SDEO. These compounds have been found in various plants and
are known to play an important role as characteristic aroma compounds of leaves, flowers or fruits of
some plants [28,41,42]. Especially, theaspirane A and theaspirane B are present in green tea, black tea,
grape and corn [43], and are believed to contribute to the unique aroma of M. tricuspidata fruit. Their
chemical structures are presented in Figure 1.
Table 1. Concentration of compounds identified in steam-distilled essential oil (SDEO) and glycosidically bound aglycone fraction (GBAF) isolated from M.
tricuspidata fruit.
Concentration (μg/100 g dw) 3)

PeakNo tR (min) Compounds RI 1) RI 2)
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SDEO GBAF
Alcohols
1 5.363 2-Methyl-1-butanol 737 1206 3.03 ± 0.25 1036.0 ± 124.6
5 7.735 trans-2-Hexen-1-ol 862 1405 7.33 ± 1.53 − 5)
8 10.318 5-Methyl-2-furfuryl alcohol 956 − 4) 3.17 ± 0.76 -
20 19.585 3,4-Dimethylcyclohexanol 6) 1109 - 15.67 ± 2.08 -
Aldehydes and ketones
3 6.644 Furfural 819 1459 53.67 ± 6.03 -
4 7.378 trans-2-Hexenal 848 1201 10.33 ± 3.06 -
2 6.284 n-Hexanal 804 1097 3.00 ± 0.80 -
7 8.777 2-Acetyl furan 903 1493 5.33 ± 1.53 -
9 10.513 5-Methylfufural 966 1508 4.13 ± 0.81 -
10 12.102 Benzaldehyde 971 1508 6.33 ± 1.53 -
11 12.491 6-Methyl-5-hepten-2-one 989 1326 6.33 ± 1.53 -
12 13.267 1-(2-Furanyl)-3-butanone 6) 1006 - 4.03 ± 0.55 -
16 15.179 Phenylacetaldehyde 1039 1629 44.33 ± 3.51 5.33 ± 1.04
18 18.958 n-Nonanal 1104 1388 140.3 ± 20.5 -
23 22.487 10-Undecenal 6) 1146 - 6.67 ± 2.52 -
24 23.306 2,4-Dimethylbenzaldehyde 6) 1158 1712 7.03 ± 1.55 -
44 41.734 Genanyl acetone 1451 1860 17.33 ± 2.52 -
52 44.505 2-Tridecanone 1493 - 23.67 ± 5.51 -
Terpenoids
36 35.657 Ylangene 1356 1464 10.93 ± 3.10 -
37 36.379 α-Copaene 1368 1477 62.33 ± 51.47 -
41 39.05 β-Caryophyllene 1409 1565 145.7 ± 10.5 -
43 39.533 α-Bergamotene 1416 1575 5.67 ± 0.58 -
45 41.982 β-Humulene 1454 - 10.33 ± 2.52 -
53 45.905 δ-Cadinene 1517 1754 147.7 ± 7.5 -
58 50.101 Caryophyllene oxide 1588 1968 56.33 ± 3.51 -
Table 1. Cont.

SDEO GBAF
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Carotenoid-derived compounds
14 15.079 2,2,6-Trimethylcyclohexanone 6) 1037 1300 5.57 ± 0.51 -
19 19.303 Isophorone 1119 1578 7.10 ± 1.85 -
22 21.727 4-Oxoisophorone 6) 1115 1674 5.33 ± 0.58 -
28 26.163 β-Cyclocitral 1214 1603 17.10 ± 1.85 -
29 28.870 β-Homocyclocitral 6) 1254 - 15.10 ± 0.85 -
31 31.253 Theaspirane A 1289 1482 121.3 ± 4.5 -
33 32.447 Theaspirane B 1306 1522 99.67 ± 9.02
42 39.454 7,8-Dihydro-α-ionone 6) 1415 1825 - 30.33 ± 2.52
48 43.389 β-Ionone 1480 1907 141.0 ± 4.4 -
49 43.637 β-Ionone epoxide 1483 1957 92.33 ± 9.71 -
55 46.692 Dihydroactinidiolide 6) 1530 2291 10.67 ± 5.51 -
59 56.486 3-Hydroxy-β-ionone 6) 1698 2646 - 160.7 ± 30.0
60 57.969 9-Hydroxymegastigma-4,6-dien-3-one (isomer #1) 6) 1705 2677 - 197.67 ± 9.45
61 58.525 4-Oxo-7,8-dihydro-β-ionol 1725 2694 - 76.00 ± 11.00
63 61.311 9-Hydroxymegastigma-4,6-dien-3-one (isomer #2) 6) 1786 2846 - 234.3 ± 24.5
Aromatic and phenolic compounds
15 15.292 Benzyl alcohol 1040 1864 - 883.7 ± 29.8
17 18.294 p-Cresol 1092 2074 393.5 ± 17.7 43.00 ± 7.55
21 19.694 2-Phenylethyl alcohol 1113 1892 - 58.85 ± 4.58
26 25.427 Pyrocatechol 7) 1203 2646 - 20.33 ± 5.51
30 31.225 Resorcinol 1288 - - 57.33 ± 10.50
32 31.523 Carvacrol 1293 2213 19.37 ± 3.46 -
34 34.006 α-Methoxy-p-cresol 7) 1331 2490 - 2783.0 ± 143.0
35 34.981 p-Vinylguaiacol 1346 2181 - 17.33 ± 3.51
25 24.925 Methyl chavicol 1171 1658 66.67 ± 9.02 -
38 37.539 2,4,6-Trihydroxybenzaldehyde 1386 - 9.33 ± 1.53 -
39 38.473 p-Hydroxybenzyl alcohol 7) 1400 2952 17.67 ± 3.06 468.1 ± 30.9
40 38.977 p-Hydroxybenzaldehyde 7) 1408 2964 - 170.0 ± 19.5
46 42.529 Tyrosol 7) 1463 2969 - 68.67 ± 4.51
47 43.524 p-Methylsalicylaldehyde 7) 1478 - 43.00 ± 10.82 4088.0 ± 147.8
50 44.116 Methyl p-hydroxybenzoate 7) 1487 1969 - 289.3 ± 12.5
51 44.439 Vanillyl alcohol 7) 1492 - - 30.67 ± 3.27
54 46.293 p-Hydroxybenzoic acid 7) 1523 - - 20.33 ± 4.51
Table 1. Cont.

SDEO GBAF
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56 46.955 Methyl caffeate 7) 1532 2593 - 31.33 ± 4.51

57 48.027 Vanillic acid 7) 1583 - - 22.67 ± 4.04
65 64.199 Methyl ferulate 1844 - - 92.00 ± 28.62
66 65.320 Ferulic acid 7) 1865 - - 383.0 ± 26.6
76 79.425 p-(p-Hydroxybenzyl)phenol 6) 2166 - - 133.1 ± 12.9
Aliphatic acids and esters
62 61.180 Myristic acid 1775 2694 124.2 ± 10.3 -
64 61.871 Ethyl myristate 1798 2041 9.33 ± 1.53 -
67 65.807 Pentadecanoic acid 1875 2822 9.33 ± 2.52 -
68 68.480 Methyl palmitate 1928 2212 55.75 ± 6.23 -
69 71.900 Palmitic acid 1986 2953 813.1 ± 39.5 -
70 72.100 Ethyl palmitate 2002 2277 291.7 ± 29.0 -
71 76.479 Methyl linoleate 2120 2485 58.16 ± 8.23 -
72 76.776 Methyl linolenate 2119 2484 55.35 ± 10.53 -
73 79.580 Linoleic acid 2169 - 363.7 ± 39.0 -
74 79.897 Linolenic acid 2175 - 176.0 ± 22.5 -
75 80.583 Ethyl linolenate 2187 2585 9.33 ± 1.53 -
Miscellaneous -
6 7.987 p-Xylene 836 1279 4.17 ± 0.76 -
13 14.526 2-Acetylthiazole 6) 1027 - 4.03 ± 0.35 -
27 25.537 2,3-Dihydrobenzofuran 1205 2381 3.77 ± 0.68 316.0 ± 29.0
1)Retention indices on DB-5MS column. 2) Retention indices on Suplecowax 10 column. 3) Values expressed as equivalents of n-decanol are given as mean ± standard deviation (n = 3).
4)Not detected or larger retention indices than 3000 in Supelcowax 10 column. 5) Not detected or less than 1.0 μg/100 g. 6) Tentatively identified based on mass spectral data only due to
lack of authentic standard compound. 7) Compounds used for antioxidant activity assays.
Foods 2019, 8, 659
Figure 1. Chemical structures of carotenoid-derived compounds identified in steam-distilled essential

oil (SDEO) and glycosidically bound aglycone fraction (GBAF) isolated from M. tricuspidata fruit.
Numbers in brackets indicate peak numbers as listed in Table 1.
In this study, the norisoprenoid compounds, 7,8-dihydro-α-ionone, 3-hydroxy-β-ionone,

4-oxo-7,8-dihydro-β-ionol and 9-hydroxymegastigma-4,6-dien-3-one (two isomers) were not detected
in fractions separated by the steam-distillation and extraction (SDE) method but in the glycosidicaly
bound volatiles fraction (GBAF). These results suggest that most of the norisoprenoid compounds
detected in the fruit are present in the form of glycosidic form rather than existing in free form in the
maturing fruit or being formed in the process of preserving the fruits after harvesting [44]. These
compounds can be derived from carotenoids by the action of related enzymes or chemical oxidation
during processing or storage of M. tricuspidata fruit. It is considered that the carotenoid is decomposed in
the process of separating volatile components by steam distillation. In particular, 3-hydroxy-β-ionone,
3,4-dihydro-α-ionone and two quantitatively detected 9-hydroxymegastigma-4,6-dien-3-one are present
in glycosidic form in some plants [45,46]. In our previous study that analyzed phenolic compounds in
the methanol extract of a fully matured fruit of the plant, we isolated a number of phenolic compounds
including quercetin and parishin derivatives [25]. In this study, only 4-Hydroxybenzyl alcohol was
able to be detected at a significant concentration suggesting most of the other phenolic compounds
must have been degraded during the steam-distillation process.
To the best of our knowledge, 13 carotenoid-derived compounds (isophorone, 4-oxoisophorone,
theaspiranes A, theaspiranes B, 7,8-dihydro-α-ionone, β-ionone, β-ionone epoxide, dihydroactinidiolide,
3-hydroxy-β-ionone, β-cyclocitral, β-homocyclocitral and two 9-hydroxymegastigma-4, 6-dien-3-one
isomers) are being identified for the first time from M. tricuspidata fruit oil. These compounds are
related to carotenoids [44]. M. tricuspidata fruit contains several carotenoids including α-carotene,
β-carotene, zeaxanthin, ruboxanthin, and lutein [47]. As described in the introduction section above,
Bajpai and colleagues have previously identified 29 compounds with 1,1-difluoro-4-vinylspiropentane,
scyllitol, 1-phenyl-1-cyclohexylethane, diethyl phthalate and 4,4-diphenyl-5-methyl-2-cyclohexenone
as major constituents in the essential oil obtained from M. tricuspidata fruit by microwave-assisted
extraction [26]. However, most of these compounds were not detected in this study. We believe that the
difference in detected components is caused by the difference in extraction method and plant samples.
In the present study, we used a fresh fruit instead of a dried one.
3.2. Chemical Composition of Glycosidically Bound Aglycone Fraction (GBAF)

It is well established that volatile components in plants and foods are present in free form while
some components exist in glycosidically bound forms [41,48,49]. The volatile components in the form
of glycoside in association with saccharides have a hydroxyl group in the molecule and are bonded
in the form of a β-glycoside. These glycosides can be hydrolyzed by β-glycosidases produced by
microorganisms to produce free form of volatiles [28,33,41]. The enzyme preparation used for such a
Foods 2019, 8, 659
purpose are enzymes with glycosidase activities such as β-d-glucosidase, α-l-arabinopyranosidase,

α-l-arabinofuranosidase and α-l-rhanosidase.
In this experiment, GBV fractions were isolated from an Amberlite XAD-2 column and then
Asp. niger cellulase was used to release aglycones from their conjugates. Compared with the gas
chromatograms of the volatile components separated by the SDE method, the number of components
detected in the GBV fraction (Supplementary Figure S1) was smaller. However, it can be clearly seen
that the intensities of the peaks are significantly higher in the GBV fraction. These results indicate that
the overall compositions of the volatile components constituting the GBV fraction are clearly different
from the volatile components present in the free form. Identities of individual compounds identified
in the SDEO and GBAF are presented in Table 1.
Regarding aldehydes and ketones which belong to the oxygenated compounds, 14 components
were detected in the volatile components fraction separated by the SDE method while only a small
amount of phenylacetaldehyde was detected in the GBAF (Table 1). These results suggest that
aldehydes and ketones present in fruits are not combined with saccharides in the form of glycosides.
In the volatile fractions separated by the SDE method, few aromatic alcohol and phenolic
compounds including constituents such as p-cresol, estragole, 2-methyl-5-(1-methylethyl) phenol,
methoxy-2-methylphenol, 2,4,6-trimethylbenzaldehyde and 2-hydroxy-4-methylbenzaldehyde were
detected in lower concentrations. By contrast, in the GBAF fraction, a large amount of aromatic alcohols
and phenolic compounds were detected. Among them, benzyl alcohol, 2-phenylethyl alcohol, resorcinol,
α-methoxy-p-cresol, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, 4-methylsalicylaldehyde,
methyl p-hydroxybenzoate, ferulic acid, methyl caffeate, pyrocatechol, p-hydroxyphenylethyl alcohol,
vanillyl alcohol, p-hydroxybenzoic acid, methyl vanillate, vanillic acid, and p-(p-hydroxybenzyl)
phenol were detected only in the GBAF (Table 1). The chemical structures of the phenolic compounds
detected in the GBAF are shown in Figure 2. As shown in the figure, one or more hydroxyl groups
are contained in the molecular structure, and thus the β-glycoside bond is hydrolyzed by treating
β-glucosidase in the presence of sugar in the form of β-glycoside in the hydroxyl group. These
compounds are smaller in molecular weight and simple in structure compared to other phenolic
compounds, but are widely distributed in plants and are known to contribute to various physiological
activities. Interesting biological activities have been reported for tyrosol, p-hydroxybenzyl alcohol
and p-hydroxybenzaldehyde including anti-oxidant activities, improving functional blood flow,
preventing memory deficits, and providing protective effects on the blood–brain barrier [50–53].
Figure 2. Cont.
Foods 2019, 8, 659
Figure 2. Chemical structures of aromatic and phenolic compounds identified in glycosidically bound
aglycone fraction (GBAF) isolated from M. tricuspidata fruit. Numbers in brackets indicate peak
numbers as listed in Table 1.
3.3. Total Phenol Contents of Fractions

The total phenol contents of the SDEO, FV and GBAF were also determined and comparisons
of the results are presented in Figure 3. Among all, the highest total phenol content was obtained
from the GBAF while the SDEO showed the lowest total phenol content (<10 mg/g dw). The total
phenol content of the FV fraction was slightly lower than the GBAF while it was much higher than
that of SDEO. The relatively higher total phenol contents in the GBAF and FV is due to the solvents
used as the efficiency of the phenolics extraction depends on the type of the solvent. During isolation
of the GBAF, extraction of the aglycones liberated by enzymatic hydrolysis employed a more polar
solvent (ethyl acetate) while only n-pentane-diethyl ether (1:1) was used in the case of SDEO. It is well
established that phenolic compounds are extracted more efficiently with polar solvents [54].
Figure 3. Total phenol contents of fractions isolated from M. tricuspidata fruit. SDEO, steam-distilled
essential oil; FV, free volatile; GBAF, glycosidically bound aglycone fraction liberated from GBV by Asp.
niger cellulose; GBV, glycosidically bound volatile fraction.
3.4. Antioxidant Activity of SDEO and GBAF

Antioxidant activities of fruit extracts have been characterized extensively [55]. In this study,
antioxidant capacities of each fraction expressed in percent of radical (DPPH and ABTS) scavenging
activities and reducing power as measured by FRAP assay, and EC50 as compared to the positive
controls BHA and BHT, are presented in Figure 4 and Table 2. In all the antioxidant property
measurement methods, the GBAF showed the highest antioxidant activity while the SDEO showed the
lowest. Considering the total yields of these fractions and their respective total phenol content results
described above, it can be said that there is a strong positive correlation between their concentrations
and their respective antioxidant activities. Maximum antioxidant activities of the GBAF were obtained
in the DPPH and FRAP methods where its activity was even higher or equivalent to those of the
synthetic antioxidants BHA and BHT. While the antioxidant properties of phenolic compounds are
Foods 2019, 8, 659
extensively demonstrated in the literature, some of the volatile compounds exclusively detected in the
GBAF might also have greatly contributed to its considerable antioxidant capacity observed in this
study. It should also be noticed that the volatile aroma components detected in higher concentrations
in the GBAF including p-Hydroxybenzyl alcohol, p-hydroxybenzaldehyde and tyrosol are well known
to have strong biological activities [56–58]. However, while antioxidant activity estimations based
on synthetic radicals are indispensable tools, many people raise concerns about their substantiation
through in vivo and clinical trials which also have more safety issues [59].
Figure 4. Antioxidant activities of steam-distilled essential oil (SDEO), free volatile (FV) and glycosidically
bound aglycone fraction (GBAF) isolated from M. tricuspidata fruit. (a) 2,2-Diphenyl-1-Picrylhydrazyl
(DPPH) free radical scavenging activity, (b) 2,2 -Azino-Bis(3-Ethylbenzothiazoline-6- Sulfonic Acid (ABTS)
free radical scavenging activity; (c) Ferric reducing antioxidant power (FRAP). Samples, 1000 ug/mL;
* Butylated hydroxyltoluene BHA, Butylated hydroxyanisole (BHT), 200 ug/mL.
Foods 2019, 8, 659
Table 2. Antioxidant activity of SDEO, FV and GBAF isolated from M. tricuspidata fruit.
Samples DPPH 1 ABTS +1 FRAP 2

SDEO 17,065.22 ± 146.27 a 1921.81 ± 49.45 a 10,638.56 ± 223.33 a
FV 2507.18 ± 24.21 b 660.72 ± 7.18 b 1963.48 ± 10.97 b
GBAF 835.33 ± 6.97 d 317.09 ± 1.99 d 529.6 ± 4.73 d
BHA 466.79 ± 7.10 e 89.15 ± 4.14 e 129.46 ± 1.61 f
BHT 535.75 ± 3.52 e 108.62 ± 1.06 e 331.26 ± 4.68 e
1 EC50 (μg/mL) values were calculated from the regression lines using six different concentrations (10–100 μg/mL) in
triplicate and data represent 50% scavenging activity. 2 Ferric-reducing antioxidant power (FRAP) were calculated
from the regression lines using six different concentrations (10–100 μg/mL in triplicate and the values were
presented by sample concentration at 0.5 of absorbance at 517 nm DPPH, 2,2-Diphenyl-1-Picrylhydrazyl; ABTS,
2,2 -Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid; SDEO, steam-distilled essential oil; FV, free volatile; GBAF,
glycosidically bound aglycone fraction liberated from glycosidically bound volatile fraction by Asp. niger cellulase.
Different superscripts in the same column indicate significant differences (p < 0.05). + , cation.
Even though antioxidant activity of the SDEO was found to be much lower than the other fractions,
it is suggested that its observed antioxidant property is related to the compounds detected in it.
Compounds such as palmitic acid, linoleic acid and p-cresol that were detected in relatively higher
concentrations in the SDEO are not important antioxidants [60,61]. Generally, the antioxidant capacity
of volatile compound fractions from M. triscuspidata fruit extracted with the SDE method and that of
GBAF are attributed to the individual components identified. The antioxidant activities expressed in
EC50 of some individual phenolic compounds evaluated in this study are also presented in Table 3.
Based on these results, it can be suggested that as most potent bioactive compounds are glycosidically
bound forms in M. triscuspidata fruit, enzymatic processing like fermentation can play an important
role in enhancing its biological activities.
Table 3. Antioxidant activity of phenolic compounds identified in GBAF.
EC50 (μg/mL)
Compounds
DPPH 1 ABTS 1 FRAP 2
Pyrocatechol 9.59 ± 1.22 e 67.68 ± 2.47 jk 74.45 ± 2.16 jk
α-Methoxy-p-cresol 1114.09 ± 114.45 d 59.55 ± 6.46 jk 3298.92 ± 126.20 f
p-Hydroxybenzyl alcohol 3357.55 ± 134.15 c 377.85 ± 4.78 f 2854.37 ± 43.04 g
p-Hydroxybenzaldehyde 1765.90 ± 364.23 d 1117.70 ± 7.01 c 7906.18 ± 60.96 c
Tyrosol 1331.74 ± 195.63 d 287.36 ± 3.70 g 92.64 ± 1.97 jk
p-Methylsalicylaldehyde 1644.14 ± 365.52 d 423.69 ± 3.13 e 19,365.27 ± 81.38 b
Methyl p-hydroxybenzoate 5241.03 ± 941.54 b 12,735.03 ± 47.26a 6789.61 ± 82.27 d
Vanillyl alcohol 27.96 ± 1.65 e 66.98 ± 1.99 jk 5928.60 ± 90.87 e
p-Hydroxybenzoic acid 10,906.51 ± 1103.69 a 6921.86 ± 50.48 b 1116.61 ± 11.69 h
Vanillic acid 48.58 ± 2.50 e 157.22 ± 4.83 h 161.18 ± 4.25 jk
Methyl caffeate 11.92 ± 0.48 e 11.91 ± 1.29 l 7.84 ± 0.28 k
Ferulic acid 24.47 ± 2.59 e 66.39 ± 2.11 jk 138.98 ± 3.73 jk
BHA 26.10 ± 0.42 e 89.27 ± 4.01 ij 129.46 ± 1.61 jk
BHT 33.71 ± 1.04 e 108.76 ± 3.93 i 331.26 ± 4.68 j
1 EC50 (μg/mL) values were calculated from the regression lines using six different concentrations (10–100 μg/mL) in
triplicate and data represent 50% scavenging activity. 2 FRAP were calculated from the regression lines curve using
six different concentrations (10–100 μg/mL) of authentic standards in triplicate and the values were presented by
sample concentration at 0.5 of absorbance at 517 nm. Different superscripts in the same column indicate significant
differences (p < 0.05).
3.5. Antioxidant Activity of Individual Phenolic Compounds in GBAF

In order to evaluate the antioxidant activities of individual compounds, EC50 of 12 compounds
identified in the GBAF was determined and the results are presented in Table 3. In all the three
assay methods, methyl caffeate displayed by far the strongest antioxidant activity expressed in EC50 .
Pyrocatechol also showed the highest DPPH scavenging activity and was even higher than the synthetic
Foods 2019, 8, 659
antioxidants BHA and BHT. As can be seen from Table 3, several phenolic compounds including
pyrocatechol, vanillyl alcohol, methyl caffeate and ferulic acid have shown antioxidant potencies higher
than that of positive controls. Ferulic acid and methyl caffeate, the two compounds that showed the
highest DPPH-scavenging activities in this study, have been previously reported to have antioxidant
activities expressed in EC50 of DPPH scavenging activity of 22 and 10.64 μg/mL for ferulic acid methyl
caffeate, respectively [50,62].
Therefore, it can be assumed that these compounds have greatly contributed to the overall higher
antioxidant activity observed in the GBAF. As described above, only a few phenolic compounds were
detected in lower concentrations in the SDEO fraction. Considering this, proper processing techniques
are required before application of M. triscuspidata fruit for its biological activity. While processing
techniques such as specific enzymatic treatments can help release some compounds, processing
methods like fermentation with microorganisms may give more efficient results. A previous study has
demonstrated an increase in the levels of phenolic compounds such as kaempferol and quercetin after
lactobacillus-mediated fermentation of M. triscuspidata leaf [63].
4. Conclusions
This study explored the chemical compositions and antioxidant activities of steam-distilled
essential oil (SDEO) and glycosidically bound aglycone fraction (GBAF) extracts from fully ripe M.
triscuspidata fruit. Thirteen carotenoid-derived compounds are being isolated for the first time in M.
triscuspidata fruit. These compounds have been associated with a variety of organoleptic properties in
other plants. A number of bioactive compounds were exclusively identified in the GBAF. It can be
suggested that the relatively higher antioxidant activity observed in this particular fraction compared
to the SDEO fraction is mainly associated with these exclusive compounds. Therefore, enzymatic
treatments of fruits suh as M. triscuspidata can significantly enhance functional properties by releasing
glycosidically bound bioactive components.
Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/8/12/659/s1,

Figure S1: Gas chromatograms of the volatile components detected in (A) steam distilled essential oil (SDEO);
(B) glycosidically bound aglycone fraction (GBAF) isolated from Maclura triscuspidata.
Author Contributions: Conceptualization & Methodology, M.-K.K. and Y.-H.K.; Investigation & Resources,
M.-K.K. and Y.-H.K.; Funding Acquisition, M.-K.K.; Formal analysis, G.-R.Y., D.-W.K., D.-H.K. and H.-A.H.;
Writing—Original Draft Preparation, Y.-H.K.; Writing—Review and Editing, Y.A.G. All authors read and approved
the final manuscript.
Funding: This work was carried out with the support of the Cooperative Research Program for Agricultural
Science & Technology Development, grant number PJ012588022019, National Academy of Agricultural Science,
Rural Development Administration, Republic of Korea.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
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foods
Article
Oleic Acid Is not the Only Relevant
Mono-Unsaturated Fatty Ester in Olive Oil
Archimede Rotondo 1, *, Giovanna Loredana La Torre 1 , Giacomo Dugo 1 , Nicola Cicero 1 ,
Antonello Santini 2 and Andrea Salvo 3
1 Department of Biomedical and Dental Sciences and Morpho-functional Imaging, University of Messina, Polo
Universitario Annunziata, Viale Annunziata, 98168 Messina, Italy; llatorre@unime.it (G.L.L.T.);
dugog@unime.it (G.D.); ncicero@unime.it (N.C.)
2 Department of Pharmacy, University of Napoli Federico II, via D. Montesano 49, 80131 Napoli, Italy;
asantini@unina.it
3 Department of Chemistry and Drug Technology, University of Roma La Sapienza, via P.le A. Moro 5, 00185
Roma, Italy; andrea.salvo@uniroma1.it
* Correspondence: arotondo@unime.it; Tel.: 39-090-676-6890
Received: 26 February 2020; Accepted: 19 March 2020; Published: 26 March 2020
Abstract: (1) Background: Extra-virgin olive oil (EVOO) is a precious and universally studied
food matrix. Recently, the quantitative chemical composition was investigated by an innovative
processing method for the nuclear magnetic resonance (NMR) experiments called Multi-Assignment
Recovered Analysis (MARA)-NMR. (2) Methods: Any EVOO 13-carbon NMR (13 C-NMR) profile
displayed inconsistent signals. This mismatch was resolved by comparing NMR data to the official
gas-chromatographic flame ionization detection (GC-FID) experiments: the analyses concerned many
EVOOs but also the “exotic” Capparis spinosa oil (CSO). (3) Results: NMR and GC-FID evidenced
the overwhelming presence of cis-vaccenic esters in the CSO and, more importantly, cis-vaccenic
13 C-NMR resonances unequivocally matched the misunderstood 13 C-NMR signals of EVOOs. The
updated assignment revealed the unexpected relevant presence of cis-vaccenic ester (around 3%) in
EVOOs; it was neglected, so far, because routine and official GC-FID profiles did not resolve oleic
and cis-vaccenic signals leading to the total quantification of both monounsaturated fatty esters. (4)
Conclusions: The rebuilt MARA-NMR and GC-FID interpretations consistently show a meaningful
presence of cis-vaccenic esters in EVOOs, whose content could be a discrimination factor featuring
specific cultivar or geographical origin. The study paves the way toward new quantification panels
and scientific research concerning vegetable oils.
Keywords: cis-vaccenic; monounsaturated fatty; glycerols; NMR analysis; olive oil; Capparis spinosa;
13 C-NMR;
MARA-NMR
1. Introduction
Extra-virgin olive oil (EVOO) comes from the supernatant phase of juice obtained after cold
pressing of Olea europaea fruits and is the fundamental dressing of any Mediterranean dish. It is
considered the liquid gold in food trading because of its crucial role in the healthy way of life model
called “Mediterranean Diet” [1]. Many scientific studies reveal that the chemical composition of
EVOO is a perfect balance leading to countless benefits for humans [2–5]. The positive biological
activities are reasonably due to the suitable presence of vegetable sterols [6], liposoluble polyphenols [7]
and other anti-oxidant hydrocarbons [8] joined to the most abundant presence of mono-unsaturated
tri-acyl-glycerol esters among the vegetable oils. Albeit the oleic ester in EVOOs is considered
the overwhelming main mono-unsaturated fatty ester so far, this work casts another important
mono-unsaturated fat potentially playing important biological roles. The wide impact of EVOOs

Foods 2020, 9, 384
composition accounts for the constantly updated European Regulation stating the chemical and taste
features, limits and official analytical techniques recognized for olive oil trade [9,10]. In the last decades,
the traditional food analysis was shocked by the nuclear magnetic resonance (NMR) as alternative
quantitative (qNMR) approach [11] flanking the officially recognized separation techniques. The
nondestructive NMR spectroscopy allows the in-situ detection of several chemical species without the
requirement of a real physical separation [8,12,13]; moreover qNMR is feasible directly or through a
clever data throughput [14,15]. The definite advantages of the NMR analyses are: a) minimal sample
treatment [8], b) simultaneous detection of a great amount of data [16], c) reduction of systematic
errors controlled by the intrinsic instrumental stability, d) constant and direct dependence between
signal integration and quantitative values because of the constant nuclear magnetic momentum for
the measured nuclei [17]. Criticism toward NMR concerned mainly sensitivity; however, it actually
depends on the machine, on sample type, on used solvent, on observed nuclei and on specific
experimental runs; this is the reason it should be evaluated from case to case [18]. After several years
of research on EVOOs composition, Rotondo et al. have developed a Multi-Assignment Recovered
Analysis (MARA-NMR) involving multi-nuclear 1 H and {1 H}13 C-NMR experiments processed by
an accustomed processing “MARA” algorithm [19]. This method successfully and quickly achieves
the quantification of many components in EVOOs samples through high-resolution spectroscopy
at 500 MHz (500 MHz HR-NMR). On the other hand, the “first” MARA-NMR scheme did not take
into account some 13 C-NMR resonances whose intensity was significant, but these were associated
to EVOO minor components (theoretically negligible and contributing for less than 1%) and, for
these studies, the best fitting goodness never reached the expected convergence. Since the official
method for the quantitative determination of glyceryl fatty esters consists in the gas-chromatographic
analysis of the corresponding methyl esters using the gas-chromatographic flame ionization detection
(GC-FID) [20], this work focused on the data comparison between NMR and GC-FID on oils in order to
solve inconsistent “leftovers” from literature. The paper evidences the neat importance of cis-vaccenic
fatty ester in EVOOs as its content is around 3%; however, it was neglected so far because, according
the official method, it is included in the level of oleic ester.
2.1. Materials and Samples

Deuterated chloroform with a small amount of Tetra-Methyl-Silane (TMS), used as internal
reference, was purchased at reagent grade from Cambridge Isotope Laboratories (CIL) Inc. Extra-virgin
olive oils were samples from awarded cultivars of different provenience representing top level food in
the seasons 2014–2015. These samples were kindly given by producers in order to carry out scientific
projects belonging to the BIOOIL program, aiming to improve knowledge about top quality products.
Some seeds were isolated from Capparis spinosa fruits (known in Sicily as “cucunci”). Afterward
seeds were dried in oven at 30 ◦ C for 2 h. The matter was grinded in a mortar until the formation of a
raw powder. This matter (20 g) was extracted in 100 mL of hexane, sonicated for 30 min at 30 ◦ C and
stirred overnight. The solution was then filtrated, and the hexane removed from the solution by using,
at first, the rotating evaporator and later N2 flow over the sample. Finally, cucunci’s seed oil (CSO)
was recovered (yield 15% w/w).
2.2. GC-FID Analysis for the Comparative Tests

Fatty acids methyl esters (FAMEs) analysis was performed according to European Union (EU)
Regulations [10]. It consists of the hydrolysis of tri-acyl-glycerides and cold transesterification with a
methanol KOH solution; in particular, the methyl esters were prepared by vigorously shaking solution
of the oil in heptane (0.1 g in 2 mL) with 0.2 mL of the methanolic KOH solution. The resulting solution
was then injected into a gas chromatograph DANI MASTER GC-FID (Milan, Italy), equipped with
a fused silica capillary column Phenomenex Zebron ZB-WAX (polar phase in polyethylene glycol)
Foods 2020, 9, 384
with a length of 30 m, internal diameter of 0.25 mm and film thickness of 0.25 μm. Helium was used
as a carrier gas at a column flow rate of 1.2 mL/min, with a split ratio of 1:100. The temperature
of the injector (split/splitless) and detector was of 220 ◦ C and 240 ◦ C, respectively. The oven was
programmed as follows: initial temperature at 130 ◦ C, final temperature at 200 ◦ C (10 min) with an
increase of 3 ◦ C/min. The fatty acid methyl esters were identified by comparing the retention times
with those of standard compounds. The relative percentage area of the fatty acids was obtained using
the following relationship: %FAX = [AX/AT] × 100, where FAX stands for fatty acids to quantify, AX is
the area of the methyl-esters and AT is the total area of the identified peaks in the chromatogram [21].
This analytical strategy is chosen for data comparison because it is officially recognized for the fatty
esters quantification, on another hand the reader should be aware that the hydrolysis-esterification
step is always tedious, laborious and time consuming, decreasing accuracy and precision. This is the
reason why, lately, alternative analytical chromatographic methods have also been proposed [22], still
showing limitations.
2.3. Sample Preparation for NMR

Sample preparation follows the same procedure successfully used by our group several years
ago [7,8,12,19]. Briefly, all the CDCl3 solutions were kept homologous by mixing 122 μL of oil and 478
μL of deuterated chloroform (CDCl3 ) into a 5 mm test-tube (EVOO or CSO in a 13.5% weight ratio). In
this study we used the same EVOOs studied in Reference 19; however, these were dissolved as different
samples and the experiments were repeated in light of the new assignments. Tubes were immediately
sealed to prevent solvent evaporation; it would affect the sample concentration influencing the chemical
shift of many signals, especially the unsaturated and carbonyl 13 C signals. These samples were readily
used for the NMR scheduled analysis so that outcomes could be suitably processed and compared to
each other.
2.4. NMR Analysis

All the samples were analyzed at a constant temperature of 298 K on a 500 MHz Avance III NMR
spectrometer endowed with a gradient assisted probe (SMARTprobe, Faellanden, Switzerland). The
shimming procedure was carried out until the field homogeneity was assessed by less than 1.5 Hz of
half-height line-width for the TMS signal.
The 1D 1 H and 13 C{1 H} NMR spectra were run at 499.74 and 125.73 MHz, respectively. This
research exploited the analytical procedure including two experiments: a) the standard 1 H experiment
with 64 scans; b) the standard 13 C NMR experiment with 32 scans. The entire procedure takes around
30 min of experimental time for any sample including preparation. Hard pulse for the maximum
sensitivity (90◦ pulse), was calibrated and constantly checked for 1 H throughout the samples being
always 8.2 ± 0.1 μs at −6 dB. 1 H-NMR experiments (type A) were run with a spectral width of 12
ppm, 64 scans, 10 s of acquisition time and 5 s of recycle delay in order to overcome problems coming
from the differences in the proton relaxation times. For the same reason the 13 C spectra (type B) were
acquired with the 90◦ hard pulse (11.2 ± 0.3 us at 6 dB) with 32 scans, 5 s of acquisition time and 20 s
for the time delay. Thanks to the MARA-NMR algorithm, these experimental elements were conveyed
together for the overall quantitative evaluation.
2.5. NMR Processing and Data Treatment

All the spectra were processed through three main software programs (ACDLab/NMR 2012
(Toronto, Ontario, Canada), MestreNova 6.6.2 (Galicia, Spain), Topspin 4.0.5 (Bruker, Milan, Italy) and
using several procedures for the coherent alignment, spectral phasing, calibration, base-line correction
and integration procedure. The best processing choices are here reported regardless the many other
adoptable procedures. Topspin processed data were selected with manual phase-correction, parametric
base-line correction with an implemented polynomial curve (for example, for experiment I absd 16
command). Calibration of experiment A was performed on the methyl group of the β-sitosterol
Foods 2020, 9, 384
signal to (δH = 0.738 ppm) with the TMS always being (δ = 0.0 ± 0.005 ppm); for 13 C calibration of
experiment B the divinyl- methylene group of the linoleate glycerols (L11; δ13C = 25.6614 ppm) was
used always keeping the known TMS 13 C signal to δ13C = 0.0 ± 0.05 ppm. The TMS calibration would
not really change the results; here, the calibration over internal signals is preferred because these are
less dependent on random conditions as explained elsewhere [19].
The serial integration of 100 regions for all the A-type experiments, and of 90 regions for experiment
B profiles, provided a pretty big matrix whose columns were the 40 studied samples EVOO and
rows represented 190 homologous integrations (see Supplementary Materials). Every column of
this matrix was processed by the mentioned MARA algorithm [19]; this theoretical architecture is
modified according to the original knowledge and assignments concerning cis-vaccenic esters (V).
The experimental coherences simply confirm the presence of a relevant amount of V, also improving
consistency assessed by low best fitting goodness (ρ) values. The extended procedure outputs up to 20
quantitative parameters [7,8,19] (Table 1) but this manuscript focuses on the 11 quantitative parameters
showing sound precision and important significance (Table 2). The data validation and experimental
error is evaluated through reproducibility (several samplings) and repeatability (analyses in different
days of the same sample).
Table 1. Abbreviations used to indicate quantitative values.
tri-acyl-glycerol percent TG%

1,2 di-acyl-glycerol percent 1,2-DG%
1,3 di-acyl-glycerol percent 1,3-DG%
squalene molecular% SQmol %
linolenate esters % Ln%
linoleates esters % L%
oleic esters % O%
palmitoleic esters % PO%
cis-vaccenic esters % V%
palmitate esters % P%
stearate esters S%
linolenate esters % in internal glyceril position Lni%
linoleates esters % in internal glyceril position Li%
oleic esters % in internal glyceril position Oi%
palmitoleic esters % in internal glyceril position POi%
cis-vaccenic esters % in internal glyceril position Vi%
palmitate esters % in internal glyceril position Pi%
sterarate esters % in internal glyceril position Si%
β-sitosterol + avenasterol + camposterol in molecular ppm VSTR
cyclo arthenol and other cyclosterols in molecular ppm CYSR
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Table 2. Quantitative data and relative deviation for 11 main variables (whose code is reported in
Table 1), as measured through Multi-Assignment Recovered Analysis-Nuclear Magnetic Resonance
(MARA-NMR) processing method working on mono dimensional 1 H and 13 C-NMR experiments for 33
samples. Standard deviations were measured through 9 different experiments on 3 identical samples
analyzed on three different days.
Sample TG% 1,2-DG% 1,3-DG% SQmol % Ln% L% O% PO% V% P% S%

S_1 96.7 ± 0.1 1.19 ± 0.06 2.1 ± 0.1 1.7 ± 0.1 0.59 ± 0.03 10.2 ± 0.1 63.9 ± 0.5 1.3 ± 0.3 2.8 ± 0.3 19.1 ± 0.3 2.2 ± 0.2
S_2 97.4 ± 0.1 1.30 ± 0.07 1.34 ± 0.09 2.2 ± 0.1 0.57 ± 0.03 12.5 ± 0.1 61.8 ± 0.4 0.9 ± 0.2 2.5 ± 0.2 19.7 ± 0.3 1.9 ± 0.2
S_3 97.7 ± 0.1 1.65 ± 0.09 0.68 ± 0.05 1.3 ± 0.1 0.51 ± 0.03 12.6 ± 0.1 59.4 ± 0.4 0.8 ± 0.2 3.7 ± 0.3 21.1 ± 0.3 2.0 ± 0.2
S_4 97.4 ± 0.1 1.39 ± 0.07 1.21 ± 0.08 0.8 ± 0.0 0.63 ± 0.03 15.4 ± 0.1 51.9 ± 0.4 1.1 ± 0.2 4.9 ± 0.4 23.7 ± 0.3 2.4 ± 0.2
S_5 97.2 ± 0.1 2.1 ± 0.1 0.70 ± 0.05 2.9 ± 0.2 0.67 ± 0.04 5.7 ± 0.1 72.8 ± 0.5 0.7 ± 0.1 1.8 ± 0.2 16.0 ± 0.2 2.4 ± 0.2
S_6 97.3 ± 0.1 1.70 ± 0.09 0.97 ± 0.07 2.7 ± 0.2 0.65 ± 0.03 2.6 ± 0.0 73.9 ± 0.5 0.7 ± 0.1 3.5 ± 0.3 16.7 ± 0.2 1.9 ± 0.2
S_7 97.3 ± 0.1 2.0 ± 0.1 0.77 ± 0.05 2.5 ± 0.2 0.61 ± 0.03 3.1 ± 0.0 74.2 ± 0.5 0.8 ± 0.2 2.7 ± 0.2 16.1 ± 0.2 2.5 ± 0.2
S_8 97.7 ± 0.1 1.59 ± 0.08 0.74 ± 0.05 2.2 ± 0.1 0.61 ± 0.03 6.4 ± 0.1 70.8 ± 0.5 0.9 ± 0.2 3.6 ± 0.3 15.5 ± 0.2 2.1 ± 0.2
S_9 96.8 ± 0.1 1.78 ± 0.09 1.4 ± 0.1 1.3 ± 0.1 0.44 ± 0.02 10.2 ± 0.1 60.0 ± 0.4 1.1 ± 0.2 5.0 ± 0.4 21.7 ± 0.3 1.6 ± 0.1
S_10 96.9 ± 0.1 1.30 ± 0.07 1.8 ± 0.1 2.3 ± 0.1 0.68 ± 0.04 7.6 ± 0.1 65.2 ± 0.5 1.1 ± 0.2 3.4 ± 0.3 20.0 ± 0.3 2.1 ± 0.2
S_11 97.4 ± 0.1 1.21 ± 0.06 1.4 ± 0.1 2.9 ± 0.2 0.61 ± 0.03 5.9 ± 0.1 67.2 ± 0.5 1.2 ± 0.3 3.6 ± 0.3 19.2 ± 0.3 2.4 ± 0.2
S_12 97.6 ± 0.1 1.32 ± 0.07 1.04 ± 0.07 3.1 ± 0.2 0.64 ± 0.03 7.7 ± 0.1 65.4 ± 0.5 2.5 ± 0.5 4.6 ± 0.4 17.4 ± 0.3 1.9 ± 0.2
S_13 97.7 ± 0.1 1.47 ± 0.08 0.88 ± 0.06 2.8 ± 0.2 0.79 ± 0.04 8.3 ± 0.1 67.1 ± 0.5 1.0 ± 0.2 2.4 ± 0.2 18.3 ± 0.3 2.0 ± 0.2
S_14 97.8 ± 0.1 1.40 ± 0.07 0.80 ± 0.06 3.4 ± 0.2 0.64 ± 0.03 4.5 ± 0.0 71.3 ± 0.5 1.2 ± 0.2 2.5 ± 0.2 17.7 ± 0.3 2.3 ± 0.2
S_15 97.8 ± 0.1 1.14 ± 0.06 1.07 ± 0.07 4.0 ± 0.3 0.60 ± 0.03 8.3 ± 0.1 64.0 ± 0.5 0.7 ± 0.1 3.1 ± 0.3 20.7 ± 0.3 2.7 ± 0.2
S_16 97.8 ± 0.1 1.48 ± 0.08 0.72 ± 0.05 3.1 ± 0.2 0.58 ± 0.03 6.2 ± 0.1 70.2 ± 0.5 0.6 ± 0.1 3.0 ± 0.3 17.5 ± 0.3 2.0 ± 0.2
S_17 97.4 ± 0.1 1.53 ± 0.08 1.08 ± 0.07 2.1 ± 0.1 0.61 ± 0.03 6.4 ± 0.1 68.4 ± 0.5 1.2 ± 0.3 3.3 ± 0.3 17.9 ± 0.3 2.2 ± 0.2
S_18 97.4 ± 0.1 1.22 ± 0.06 1.4 ± 0.1 2.3 ± 0.1 0.58 ± 0.03 6.7 ± 0.1 68.5 ± 0.5 0.8 ± 0.2 2.5 ± 0.2 19.1 ± 0.3 1.7 ± 0.1
S_19 97.6 ± 0.1 1.43 ± 0.07 0.98 ± 0.07 3.5 ± 0.2 0.79 ± 0.04 7.5 ± 0.1 65.6 ± 0.5 0.7 ± 0.2 2.8 ± 0.3 20.7 ± 0.3 1.9 ± 0.2
S_20 97.2 ± 0.1 1.59 ± 0.08 1.17 ± 0.08 2.0 ± 0.1 0.63 ± 0.03 9.3 ± 0.1 64.8 ± 0.5 1.1 ± 0.2 3.7 ± 0.3 19.0 ± 0.3 1.5 ± 0.1
S_21 97.2 ± 0.1 1.70 ± 0.09 1.10 ± 0.08 3.0 ± 0.2 0.70 ± 0.04 7.6 ± 0.1 66.2 ± 0.5 0.6 ± 0.1 3.1 ± 0.3 19.9 ± 0.3 1.9 ± 0.2
S_22 97.7 ± 0.1 1.49 ± 0.08 0.78 ± 0.05 3.6 ± 0.2 0.73 ± 0.04 7.5 ± 0.1 64.8 ± 0.5 1.3 ± 0.3 3.4 ± 0.3 20.7 ± 0.3 1.7 ± 0.1
S_23 98.0 ± 0.1 1.63 ± 0.09 0.37 ± 0.03 3.4 ± 0.2 0.76 ± 0.04 6.9 ± 0.1 65.4 ± 0.5 1.4 ± 0.3 2.8 ± 0.3 20.1 ± 0.3 2.5 ± 0.2
S_24 97.6 ± 0.1 1.58 ± 0.08 0.85 ± 0.06 3.4 ± 0.2 0.73 ± 0.04 7.9 ± 0.1 65.9 ± 0.5 1.1 ± 0.2 2.5 ± 0.2 20.0 ± 0.3 2.0 ± 0.2
S_25 98.0 ± 0.1 1.21 ± 0.06 0.78 ± 0.05 2.4 ± 0.2 0.58 ± 0.03 7.2 ± 0.1 66.9 ± 0.5 0.6 ± 0.1 3.6 ± 0.3 19.2 ± 0.3 2.0 ± 0.2
S_26 97.3 ± 0.1 1.27 ± 0.07 1.4 ± 0.1 2.0 ± 0.1 0.68 ± 0.04 4.4 ± 0.0 73.3 ± 0.5 1.3 ± 0.3 3.0 ± 0.3 15.6 ± 0.2 1.8 ± 0.1
S_27 97.4 ± 0.1 1.48 ± 0.08 1.13 ± 0.08 2.5 ± 0.2 0.64 ± 0.03 4.4 ± 0.0 72.2 ± 0.5 0.9 ± 0.2 2.6 ± 0.2 16.9 ± 0.2 2.3 ± 0.2
S_28 97.8 ± 0.1 1.31 ± 0.07 0.85 ± 0.06 2.5 ± 0.2 0.64 ± 0.03 7.1 ± 0.1 73.9 ± 0.5 1.3 ± 0.3 1.5 ± 0.1 13.7 ± 0.2 1.8 ± 0.1
S_29 96.9 ± 0.1 1.45 ± 0.08 1.6 ± 0.1 2.7 ± 0.2 0.65 ± 0.04 5.2 ± 0.0 70.5 ± 0.5 1.4 ± 0.3 2.6 ± 0.2 17.1 ± 0.2 2.4 ± 0.2
S_30 97.9 ± 0.1 1.22 ± 0.06 0.89 ± 0.06 1.9 ± 0.1 0.64 ± 0.03 4.8 ± 0.0 73.1 ± 0.5 0.9 ± 0.2 2.9 ± 0.3 15.4 ± 0.2 2.3 ± 0.2
S_31 96.9 ± 0.1 2.5 ± 0.1 0.50 ± 0.03 2.6 ± 0.2 0.56 ± 0.03 6.3 ± 0.1 64.5 ± 0.5 0.5 ± 0.1 5.2 ± 0.5 21.5 ± 0.3 1.4 ± 0.1
S_32 96.0 ± 0.1 3.0 ± 0.2 0.99 ± 0.07 3.1 ± 0.2 0.69 ± 0.04 9.4 ± 0.1 61.8 ± 0.4 1.0 ± 0.2 4.4 ± 0.4 21.1 ± 0.3 1.8 ± 0.1
S_33 95.8 ± 0.1 2.3 ± 0.1 1.9 ± 0.1 2.2 ± 0.1 0.66 ± 0.04 8.7 ± 0.1 65.6 ± 0.5 1.0 ± 0.2 3.9 ± 0.4 18.3 ± 0.3 1.9 ± 0.1
2.6. Mathematical Background of MARA-NMR and Updates

The used algorithm MARA-NMR was invented in this laboratory, exploiting the very simple idea
that all NMR signals rise from active nuclei that belong to compounds and contribute according to: a)
relative concentration, b) number of resonating nuclei, c) possible overlaps with homologous nuclei
maybe belonging to other compounds [19]. If this theoretical statement and a suitable assignment is
correct, the experimental profile should perfectly match our theoretical reconstruction. As explained in
the original paper [18] experimental data are not ideal data-points, however we have designed this
algorithm able to optimize quantitative parameters in order to minimize the overall deviations between
experimental and theoretical outcomes enclosed in the function ρ which is the best-fitting goodness.
xf n ◦ 2
γxj Ixj i=a N NUCi ∗Ci
ρ= ωxj − . (1)
xj=x1 Iref N◦ NUCref ∗Cref
The intensity of any signal in the spectrum Ixj respect to a reference signal Iref should even out the
relative concentration (Ci against Cref ) of the magnetically active nuclei NUCi actually assigned to that
Foods 2020, 9, 384
signal. Coefficients ω and γ are empirical parameters able to reduce experimental deviation improving
the algorithm; theoretically speaking the best fitting goodness ρ should be 0 but in the real world we
accept low values. The introduction of 18 new assignments for the cis-vaccenic ester, by enhancing just
one quantitative parameter referred to the “new” component greatly lowered the best fitting goodness
giving the proof of concept about the assignment. The 20 quantitative parameters are derived by 11
expressions derived from A experiments and 65 expressions derived from B experiments put together
in the same expression as equation (1) containing 76 xj members and 20 i compounds. In order to
preserve the quantitative proportion of 13 C integrations, despite the uneven nOe relayed on total
decoupled carbon nuclei, adopted equations in the sum (1) are divided in blocks of nuclei with the same
chemical environment (methyl terminal carbons, methylene inner chain carbons, vynil-methylene, etc.).
It is demonstrated that MARA-NMR keeps the quantitative information as reported in Supplementary
Materials and in Reference [19].
3. Results
Figure 1 shows the chemical moieties, related abbreviations and the adopted labelling scheme;
Figure 2 reports the GC-FID profile referring to the CSO extracted in our laboratory and Figure 3
represents the 13 C-NMR profile of EVOO and CSO in the unsaturated region (127–131 ppm) along
with the relative assignment witnessing the presence of the cis-vaccenic ester. As easily foreseeable,
other NMR spectral regions also clearly showed cis-vaccenic resonances; however, a total assignment
of 18 13 C carbon atoms was challenged by the many overlaps. Previous pioneering studies pointed out
the challenging quantitative decoding of the mono-unsaturated fatty esters mixture in EVOOs [23].
Specifically, other minor mono-unsaturated fatty esters (MUFE) were taken into account; beyond the
oleic (O) are also considered cis-vaccenic (V), eicosenoic (E) and palmitoleic (PO) [24,25]. On the other
hand, data coming from known EVOOs compositions, limit the quantitative contribution of E and PO
below 1% [9] and it is consistently witnessed by the lack of defined resonances in the regions where
these esters should not have overlap with other similar constructs. The Multiple Assignment Recovered
Analysis (MARA-NMR) takes advantage of any spectral section also overcoming the overlap issues
hampering, so far, the independent quantification of mono-unsaturated fatty esters. Specifically, in
this case, MARA-NMR processing definitely led to the detection and quantification of the V esters
(consistently all over the recorded spectral span). Among the 20 variables feed out from MARA-NMR
whose code is reported in Table 1, we herein have restricted our considerations to the most meaningful
11 variables reported in Table 2 along with the relative standard deviation.
With respect to the other studies [24,25] the new information remarkably smooths discrepancies
between 1 H and 13 C-NMR as the mono-unsaturated fatty esters contribution in 1 H-NMR matches the
contribution of O and V esters, which actually should be also somewhat enhanced by the minor PO
and E esters’ contribution. Because of the tricky GC-FID resolution between V and O, also referred to
in the European regulation (which suggests to report the whole V+O contribution), it is not always
possible to compare GC and NMR data. However, the new available data, display the best fitting so far
obtainable (Figure 4) concerning the measurements of mono-unsaturated (O + V + PO), saturated (P +
S), di-unsaturated (L) and tri-unsaturated (Ln) fatty esters. The average V contribution is around 3%
and it is consistent with previous NMR [23] and GC-FID [26] analyses; on the other hand, we think
that MARA-NMR is the most versatile method suitable for serial processing of several samples and
data. We think that this remarkable parameter in EVOOs cannot be ignored, since it is not constant by
shifting from sample to sample, therefore it could assess specific features of different food products.
The V quantification is not a marker for this study according to Table 2; however, it will trigger many
important statistical considerations.
Foods 2020, 9, 384
Figure 1. Chemical scheme of the fatty esters commonly found in olive oils with relative abbreviation.
Usually these acyl residues are esters of the glycerol moiety. The labelling scheme of carbon atoms is
adopted in this paper for assignments and discussion.
This enlightened an important piece of information concerning the cis-vaccenic ester as main
compound in CSO but also as relevant ester contributing to the EVOO mixture. This last element
was incredibly ignored so far. Table S1 (Supplementary Materials) reports the extended panel of 20
quantitative variables considered in the study for 33 samples (see details in Supplementary Materials).
These values are obtained by MARA-NMR—a post-processing algorithm working over the two
experiments A and B type.
Foods 2020, 9, 384
Figure 2. Expanded region of interest in the gas-chromatographic flame ionization detection (GC-FID)
profile for Capparis spinosa oil; oleic (O) and cis-vaccenic (V) methyl esters are resolved for the
quantification. In the case of extra-virgin oil the O peak is around 20 times more than V. Other labelled
signals are linolenic (Ln), linoleic (L) and stearic (S) esters
Figure 3. 13 C-NMR profiles for olive oil (EVOO) in gray and capparis seed oil (CSO) in black. All the
assignments for oleic (O), linoleic (L) linolenic (Ln) and cis-vaccenic (V), with the number representing
carbon atom position respect to the 1 carboxyl position, are pretty known and coherent with quantitative
and literature data.
Foods 2020, 9, 384
Figure 4. Comparison between MARA-NMR and GC-FID measured quantitative parameters referred
to: (A) mono-unsaturated (MUFA), (B) saturated (SFA), (C) Linoleic (L) and (D) Linolenic (Ln) esters in
relative percent ratio.
4. Discussion
The previously reported assignments for EVOOs 13 C-NMR definitely accounted for five fatty esters
in the following quantitative order: oleic (O), palmitic (P), linoleic (L), stearic (S) and linolenic (Ln) [27,28].
Some other tentative assignments concerned mono-unsaturated fatty esters like palmitoleic (PO) and
11-eicosenoic (E) constructs [29]. Despite the wide availability of NMR reports [30], none of these clearly
explained the systematic presence of unknown resonances (in our processing batch at 129.92, 129.82,
31.82, 22.69 ppm and others) which account for a relevant quantitative contribution (around 3%, Figure 3).
Scientific hesitancy probably owes to the general opinion that the total amount of other fatty esters is
limited to less than 1% of EVOOs. This idea was questioning the NMR technique itself as possible
analytical method but the serendipitous extraction of the Capparis spinosa oil (CSO) allowed us to solve
this inconsistency because of the remarkable presence of cis-vaccenic (V) esters. The comparison between
NMR and GC-FID analyses of CSO consistently confirmed the main presence of the V ester with a minor
contribution of the O ester. The analogous analytical approach executed over several EVOO samples
made us realize that the detected mono-unsaturated fatty esters were again O and V but in a reversed
quantitative proportion respect to the CSO. Against this background, the main NMR resonances attributed
to V in other peculiar food matter [31–33] (as also the reported CSO sample) were matching the EVOO
signals as reported in Figure 3. It definitely gave us the chance to include the V component in the EVOO
quantitative panel according to the 13 C-NMR resonances afore mentioned. The V remarkable presence
is not just a production side product as we did not observe the presence of trans isomers (resonances
downfield respect 5.40 ppm in the 1 H-NMR and relative other singlets in the 13 C-NMR). Once again these
results confirm the stability and sound presence of the cis form of unsaturated esters in spite of the minor
thermodynamic stability. In order to perform the updated comprehensive quantitative NMR analysis of
EVOO we have adopted an accustomed procedure based on MARA-NMR. Although it is not the first
analytical comparison between GC and NMR [34,35], the novel MARA-NMR strategy suitably refined
according to the new information led to a very good fitting (Figure 4). The whole outcome is reported
in Tables (Tables 1 and 2, and Tables S1 and S2 in Supplementary Data). In order to get consistent data,
we have chosen to compare the percent presence of L and Ln as detected, whereas the saturated fatty
esters (SFA%) were considered as the sum S+P and the mono-unsaturated fatty esters (MUFA) were
Foods 2020, 9, 384
considered as O+V+PO. We think it is actually an important parallel evaluation whose general trend shows
a very good fitting also kept with samples showing sensibly different proportions. Finally, by properly
considering all the mono-unsaturated fatty esters, the MUFA% estimation reached an unprecedented very
good matching. On the other hand, the slight systematic overestimation of GC-FID respect to the NMR
for L% and Ln% and underestimation of SFA% deserves to be elucidated with further studies requiring
standard mixtures similar to EVOO, which is a tri-acyl-glycerol mixture. At the moment, these substrates
are not available but work is in progress to develop further information. Although it is not the first
case of V detection and also quantification [36], the EVOOs routine quantifications barely evidence the
resolution for O-V peaks; this is clearly shown in the GC picture of the European Regulation 2013 [9]. Our
observations also demonstrated that new GC-FID columns keep a better (affordable) resolution, whereas
routine instruments adopted for serial records easily present the V peak as O shoulder. Fortunately,
recorded 13 C-NMR provide the missing information about the V fraction (not really taken into account so
far) for any EVOO sample (Figure 3). According to our opinion, future studies could take advantage from
a “powered” MARA-NMR working over sensitivity-enhanced 13 C-NMR profile (optimized scans); these
could push further the frontiers of quick qNMR in EVOOs by enabling the independent quantification of
fatty esters in the 2- internal position of glycerides but also the improved quantification of other minor
components (see Supplementary Materials). This contribution also opens the way toward new studies
concerning sensory attributes, geographical origin and beneficial effects [37] of EVOO as fundamental
functional food with the major presence of glycerol esters [38].
5. Conclusions
This study definitely assesses the constant and relevant presence in olive oils of a not-oleic
mono-unsaturated fatty ester called cis-vaccenic ester. It resolves the literature controversies concerning
the assignment of some 13 C-NMR resonances but, more importantly, it brings back the expected
coherency between NMR and chromatography data. The serendipitous comparison of GC-FID and
NMR profiles for the “exotic” Capparis spinosa oil evidenced the overwhelming amount of cis-vaccenic
ester in this matrix but also unambiguously confirmed 13 C-NMR assignments also validated in olive
oil. By reconsidering the NMR and GC-FID of olive oils, it turned out the surprising quantitative
contribution (around 3%) of cis-vaccenic ester. The official GC method does not always perform the
required resolution to resolve and quantify oleic and cis-vaccenic esters and this is leading to the
undistinguished quantification of both mono-unsaturated fatty esters. It opens up great potential
for any technique able to clearly resolve cis-vaccenic moieties (just like 13 C-NMR) in the study of
extra-virgin olive oils.

Table S1: Analyzed samples coming from awarded BIOOIL competition 2014. The used code is connected to the
provenance and to the known cultivar. Table S2: Quantitative data and relative deviation for 20 main variables, as
measured through MARA-NMR processing method working on mono dimensional 1 H and 13 C-NMR experiments
for 33 samples. Table S3: General scheme of MARA-NMR referred just to the first sample. There are several
blocks: namely 1 H-NMR integrations with assignment (100 entries), DPFGSE 1 H-NMR integrations (17 entries,
not used in this study), 13 C-NMR integrations (90 entries) along with some sum of integrals belonging to the same
spectral block. Where possible, assignments are performed respecting the chemical position indicated also in
other studies about the NMR of olive oil compounds (see Figure 1), for the fatty esters the abbreviation is followed
by a number indicating the distance from the carboxyl position (generally from 1 to 18). These first rows will be
used in the following equations according to the style of (1) (see main text) conveyed as square sum in the raw
called RHO. The RHO value is minimized playing around with the quantitative variables so that the theoretical
outcome is best-fitting the real (independent) variables, Figure S1. Stack-plot of eight olive oils coming from Sicily.
The reported assignment follows the labeling used in the main manuscript (scheme 1). The expanded regions
around 22 and 32 ppm show the clear presence of cis-vaccenic acid signals useful for the quantification within
MARA-NMR quantification. The aromatic region is already reported in Figure 2 of the main text.
Author Contributions: Conceptualization, A.R. and A.S. (Andrea Salvo); methodology, A.R.; software, A.R.;
validation, A.R., G.L.L.T. and A.S. (Andrea Salvo); formal analysis, A.R., G.L.L.T.; investigation, A.R.; data curation,
A.R., A.S. (Andrea Salvo) and G.L.L.T.; writing—original draft preparation, A.R.; writing—review and editing,
A.R and G.L.L.T.; visualization and supervision, A.R., A.S. (Antonello Santini), A.S. (Andrea Salvo) and G.D.;
Foods 2020, 9, 384
background, N.C. and A.S. (Antonello Santini). All authors have read and agreed to the published version of
the manuscript.
Acknowledgments: Once again we thank the institution “University of Messina,” which, in spite of poor means
and the difficult situation of the south Italian research, does not give up.
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foods
Article
Bioactivity-Guided Fractionation and NMR-Based
Identification of the Immunomodulatory Isoflavone
from the Roots of Uraria crinita (L.) Desv. ex DC
Ping-Chen Tu 1 , Chih-Ju Chan 2 , Yi-Chen Liu 3 , Yueh-Hsiung Kuo 2,4,5 , Ming-Kuem Lin 2, * and
Meng-Shiou Lee 2, *
1 Program for Cancer Biology and Drug Discovery, China Medical University and Academia Sinica,
Taichung 404, Taiwan; pingchen.tu@gmail.com
2 Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University,
Taichung 404, Taiwan; ruby24301@gmail.com (C.-J.C.); kuoyh@mail.cmu.edu.tw (Y.-H.K.)
3 Institute of Biomedical Science and Rong Hsing Research Center for Translational Medicine, National
Chung-Hsing University, Taichung 402, Taiwan; sealioler@gmail.com
4 Department of Biotechnology, Asia University, Taichung 413, Taiwan
5 Chinese Medicine Research Center, China Medical University, Taichung 404, Taiwan
* Correspondence: linmk@mail.cmu.edu.tw (M.-K.L.); leemengshiou@mail.cmu.edu.tw (M.-S.L.);
Tel.: +886-4-2205-3366 (M.-K.L.); +886-4-2205-3366 (M.-S.L.)
Received: 7 August 2019; Accepted: 10 October 2019; Published: 3 November 2019
Abstract: Uraria crinita is used as a functional food ingredient. Little is known about the association
between its immunomodulatory activity and its metabolites. We applied a precise strategy
for screening metabolites using immunomodulatory fractions from a U. crinata root methanolic
extract (UCME) in combination with bioactivity-guided fractionation and NMR-based identification.
The fractions from UCME were evaluated in terms of their inhibitory activity against the production
of pro-inflammatory cytokines (IL-6 and TNF-α) by lipopolysaccharide (LPS)-stimulated mouse
bone marrow-derived dendritic cells (BMDC). The role of the isoflavone genistein was indicated
by the 1 H NMR profiling of immunomodulatory subfractions (D-4 and D-5) and supported by the
result that genistein-knockout subfractions (D-4 w/o and D-5 w/o) had a lower inhibitory activity
compared to genistein-containing subfractions. This study suggests that genistein contributes to the
immunomodulatory activity of UCME and will help in the standardization of functional food.
Keywords: Uraria crinita; isoflavone; genistein; NMR-based identification; dendritic cells
1. Introduction
Uraria crinita (L.) Desv. ex DC. (UC) belongs to the family of Leguminosae. It is a popular
and commercially important medicinal plant, distributed widely in Taiwan and cultivated mostly
in Mingjian Township, Nantou (approximately 20–60 hectares per year). UC roots, also known as
“Taiwanese ginseng” due to the similar potency and aroma of its decoction and ginseng, have been
traditionally used to coordinate the gastrointestinal system, thanks to their detumescent and antipyretic
effects, indicating immunomodulatory activity [1]. UC roots are used as dietary supplements for
treating childhood skeletal dysplasia. UC roots have therefore been developed as valuable and
commercial functional food in Taiwan. This herb has been shown to have antioxidant [2] and
antidiabetic activities [3] and the potential to stimulate bone formation and regeneration [4]. Previous
phytochemical investigations on UC roots led to the isolation of fatty acids, steroids, triterpenoids,
phenolics, lignans, flavonoids, and isoflavonoids [2,4–6]. However, little is known about the association
between the immunomodulatory activity and the metabolites in this herb.

Foods 2019, 8, 543
Dendritic cells (DCs), acting as antigen-presenting cells (APCs), are the major leukocytes, with a
critical role in regulating adaptive immune responses [7]. Immature DCs, characterized by a high
antigen uptake ability and poor antigen-presenting function, reside in the peripheral tissues, where
they regularly uptake and process self-antigens and maintain self-tolerance [7]. Upon activation,
immature DCs undergo maturation and migrate to adjacent lymph nodes or to the lymph organs,
after the recognition of pathogen-associated molecular patterns and damage-associated molecular
patterns by pattern recognition receptors, mostly Toll-like receptors (TLRs) [8]. This process is
accompanied by the upregulation of the expression of major histocompatibility complex (MHC) class
II molecules and several co-stimulatory molecules (CD40, CD80, and CD86) on the surface of cells [9].
Mature DCs generate more pro-inflammatory cytokines (TNF-α, IL-6, and IL-12) required for T cell
activation and have the ability to present antigens to T cells, linking the innate and adaptive immune
systems [9]. Therefore, targeting DCs is a promising strategy for immunomodulation. Medicinal herbs,
which modulate the function of DCs, can potentially be developed into botanical drugs for treating
immune disorders.
The promising potential of the herbal industry can only be achieved through the standardization
of the composition of herbal products and the assurance of proper quality control [10]. However,
the variable constituents of herbal products, owing to genetic, cultural, and environmental factors,
have made the quality of herbal medicines difficult to control. Nuclear magnetic resonance (NMR) has
been demonstrated as one of the best analytical approaches to identifying metabolites and providing
both qualitative and quantitative information [11]. While NMR suffers from a relatively poor sensitivity
compared to mass spectrometry (MS), it has some unique advantages over MS-based approaches,
including a non-destructive nature, high robustness, high reproducibility, high reliability, and powerful
ability to provide structural information for unknown metabolites [12]. NMR spectroscopy could offer
structural elucidation, achieved by the chemical shift, multiplicity, coupling constant, and integration
of (primary or secondary) metabolite signals in crude extracts. Additionally, the 1 H NMR signal
intensity, proportional to the relative number of protons, could provide useful information about the
quantity of the different metabolites in herbal extracts. Therefore, NMR has been successfully used for
fingerprinting and the metabolite discrimination of herbs [13,14]. Currently, NMR-based metabolomic
analysis is widely used in studies of nutrients, environment, plant physiology, drug metabolism,
toxicology, as well as for diagnoses and for the quality control of herbal products [10,15,16].
In this study, a combination of bioactivity-guided fractionation and NMR-based identification
led to the elucidation of the central role of the immunomodulatory isoflavone genistein present in
UC root methanolic extract (UCME) against the activation and maturation of lipopolysaccharide
(LPS)-stimulated DCs. This strategy could prevent unnecessary time-consuming isolation procedures
and provide a rapid tool for the identification of the active ingredients of herbs. Our findings suggest
that UC roots can be applied as an immunosuppressive functional food, of which genistein can be a
chemical marker for quality control. The standardization of UC roots could therefore be improved.
2.1. The EtOAc-Soluble Fraction from UCME Inhibited LPS-Stimulated DC Activation

The immunomodulatory effects of UC roots on DCs have not been reported. Here,
the Gram-negative bacterial endotoxin LPS was used to stimulate bone marrow-derived dendritic
cell (BMDC) activation, as a model for investigating the immunomodulatory effects of UC roots on
DCs. First, the immunomodulatory effects of UCME and its EtOAc-, n-BuOH-, and H2 O-soluble
fractions were evaluated against the production of pro-inflammatory cytokines, including TNF-α
and IL-6, which is a hallmark of DC activation. As shown in Figure 1A, LPS-stimulated BMDC
activation was suppressed by UCME, and UCME ability to inhibit DC activation was mainly associated
with its EtOAc-soluble fraction. In addition, the treatment with UCME and its various partitioned
fractions, at concentrations below 100 μg/mL, did not exhibit any cytotoxicity in BMDC (data not
Foods 2019, 8, 543
shown). In summary, our results revealed that the EtOAc-soluble fraction of UCME may contain
immunomodulatory phytochemicals which attenuate the activity of DCs.
Figure 1. The effects of UC methanolic extract and of its EtOAc-, n-BuOH-, H2 O-soluble fractions and
subfractions of the EtOAc-soluble fraction on pro-inflammatory cytokine production in LPS-stimulated
dendritic cells (DCs). DCs were untreated or treated with LPS (100 ng/mL, white bar). (A) Methanolic
extract and EtOAc, n-BuOH, and H2 O subfractions (25 μg/mL, gray bar, or 100 μg/mL, black bar) were
used. (B) EtOAc and Fr. A to I subfractions (50 μg/mL, black bar) were used. (C) Fr. D and D-1 to D-6
subfractions (10 μg/mL, hatch bar, 25 μg/mL, gray bar, or 50 μg/mL, black bar) were used. Supernatants
were collected 6 h after the treatment. The production of cytokines (TNF-α and IL-6) was measured by
ELISA. The data shown are the mean ± SD of three independent experiments; ### p < 0.001; * p < 0.05;
** p < 0.01; *** p < 0.001 (Scheffe’s test) for comparisons of the treated and untreated LPS-stimulated
DC samples.
2.2. Bioactivity-Guided Fractionation and NMR-Based Identification of the EtOAc-Soluble Fraction of UCME
The EtOAc-soluble fraction of UCME was subjected to silica gel column chromatography
(EtOAc/n-hexane/MeOH, gradient), and each collected fraction was analyzed by thin-layer
chromatography (TLC) and assign to one of nine main fractions (Fr. A to I, Figure 2).
Foods 2019, 8, 543
Figure 2. Bioactivity-guided fractionation and NMR-based identification of genistein from the roots of
Uraria crinita (UC). * indicates the most potent subfractions or constituents against pro-inflammatory
cytokine production in lipopolysaccharide (LPS)-stimulated DCs. UCME: UC root methanolic extract,
BMDCs: bone marrow-derived dendritic cells.
Foods 2019, 8, 543
Among them, fraction D significantly inhibited the production of TNF-α and IL-6 in BMDC
(Figure 1B). Furthermore, the subfractions D-4 and D-5 from fraction D indicated the most potent
inhibitory effects against DC activation (Figure 1C).
In order to elucidate the association between bioactivity and metabolites in subfractions D-1 to D-6,
1 H NMR spectroscopy was conducted (Figure 3). This could offer structural elucidation, achieved by
the chemical shift, multiplicity, coupling constant, and integration of metabolite signals in the mixtures.
Figure 3. Selected 1 H NMR profiling (acetone-d6 , 500 MHz) of subfractions D-1 to D-6. * indicates
the characteristic singlet signals, δH 8.13, of isoflavones; ↓ indicates the 1 H spectral data of genistein:
δH 7.44 (2H, d, J = 8.7 Hz), 6.88–6.94 (overlaps), 6.40 (1H, d, J = 2.2 Hz), and 6.28 (1H, d, J = 2.2 Hz).
According to the 1 H NMR profiling of subfractions D-1 to D-6, the characteristic singlet signals
(δH 8.13) indicated the presence of isoflavonoids in D-3, D-4, D-5, and D-6. The inhibitory activity
of subfractions D-4 and D-5 was then related to the additional signals [δH 7.44 (2H, d, J = 8.7 Hz),
6.88–6.94 (overlaps), 6.40 (1H, d, J = 2.2 Hz), and 6.28 (1H, d, J = 2.2 Hz)], which were not visible
for subfractions D-3 and D-6. A pair of aromatic protons with a meta coupling (J = 2.2 Hz) indicated
the presence of a 1,3,4,5-tetrasubstituted aromatic ring. An aromatic proton signal at δH 7.44 (2H, d,
J = 8.7 Hz) revealed that the other proton signal might be overlapping at δH 6.88–6.94 ppm, suggestive
of a 1,4-disubstituted aromatic ring. To determine the overlapping peaks at δH 6.88–6.94 ppm, the 13 C
NMR and 2D NMR experiments (Figures S1–S3), including heteronuclear single-quantum correlation
(HSQC) and heteronuclear multiple-bond correlation (HMBC), were then conducted. On the basis of
these results, genistein was identified [17] from the genistein-containing subfractions (D-4 and D-5).
Bioactive fractions, together with the active ingredient, were rapidly obtained from the combination of
bioactivity-guided fractionation and NMR-based identification of the UCME EtOAc-soluble fraction.
Foods 2019, 8, 543
2.3. The Role of Genistein in Modulating LPS-Stimulated DC Activation of UCME

To clarify the role of genistein in modulating LPS-stimulated DC activation, the genistein-containing
subfractions D-4 and D-5 were further isolated by normal-phase semipreparative HPLC to isolate
genistein (24 mg/1.136 kg dry material) and genistein-knockout subfractions (D-4 w/o and D-5 w/o,
Figure S4). The yield of genistein was 15 times higher than that achieved in a previous study [6]. These
subfractions were then evaluated in terms of their inhibitory activity by measuring cytokine production
in LPS-stimulated DCs. As shown in Figure 4A, the genistein-containing subfractions (D-4 and D-5)
significantly suppressed the production of cytokines (IL-6 and TNF-α), while the genistein-knockout
subfractions D-4 w/o and D-5 w/o showed lower inhibitory activity. As illustrated in Figure 4B,
concerning TNF-α production, the inhibition percentage of the genistein-knockout subfraction D-4 w/o
(9% at 25 μg/mL and 18% at 50 μg/mL) was dramatically lower than that of the genistein-containing
subfraction D-4 (35% at 25 μg/mL and 55% at 50 μg/mL).
Figure 4. The effects of the subfractions, containing or not containing genistein, on the production of
pro-inflammatory cytokines in LPS-stimulated DCs. (A) DCs were untreated or treated with LPS (100
ng/mL, white bar), LPS + genistein-containing subfractions, or LPS + genistein-knockout subfractions
(25 μg/mL, gray bar or 50 μg/mL, black bar), as indicated. Supernatants were collected 6 h after the
treatment. The production of cytokines (TNF-α and IL-6) was measured by ELISA. The data shown are
the mean ± SD of three independent experiments. ### p < 0.001; * p < 0.05; ** p < 0.01; *** p < 0.001
(Scheffe’s test) for comparisons of the treated and untreated LPS-stimulated DCs. (B) The inhibition
percentage (%) of the subfractions, with and without genistein, of cytokine production was derived
from the data in (A).
In addition to the identification of genistein, the fractionation and HPLC isolation of UCME
identified eight compounds, including an isoflavone (lupinalbin A [18]), three phenolic acids
(p-hydroxybenzoic acid [19], salicylic acid [20], and vanillic acid [19]), two fatty acids (monomethyl
succinate [21] and 1,10-decanedioic acid [22]), and two steroids (a mixture of β-sitosterol and
stigmasterol [23]). Their structures (Figure 5) were identified by comparing their spectroscopic
data with data in the literature. Among the isolates, lupinalbin A, p-hydroxybenzoic acid, vanillic
Foods 2019, 8, 543
acid, monomethyl succinate, and monomethyl succinate were isolated from this herb for the first time.
All isolates were also assessed in terms of their inhibitory effect on DC activation. However, only the
analogue of genistein, lupinalbin A, showed a moderate inhibitory activity against LPS-stimulated DC
activation (Figures S5 and S6).
Figure 5. Chemical structures of the compounds identified in this study.
1 H NMR signal intensity is absolutely proportional to the relative number of protons. Therefore,
the relative quantity of the different metabolites could easily be observed in the 1 H NMR spectra
(Figure 3). The major metabolites (identified by signals with a stronger intensity) in subfractions
D-4 and D-5 are shown in Table 1. Interestingly, the 1 H NMR profiling of subfractions D-4 and
D-5 revealed the presence of other unknown genistein derivatives, which might contribute slightly
to the immunomodulatory effects, because of their low abundance or poor activity. Additionally,
the well-known bioactive isoflavonoids, including daidzein, formononetin, equol, and glycitein, were
not present as major metabolites in fraction D. Therefore, genistein was suggested as having the central
role in the modulation by UCME of LPS-stimulated DC activation.
2.4. LPS-Stimulated DC Maturation was Impaired by Genistein at Non-Cytotoxic Concentrations

To exclude the possibility that genistein caused cytotoxicity, the cell viability of DCs was determined
via the CCK8 assay. As shown in Figure 6, genistein induced a significant level of DC death at 40 μM.
Thus, the significant inhibition of cytokine production by 40 μM genistein should be attributed to
its cytotoxicity in DCs. However, the LPS-stimulated production of pro-inflammatory cytokines
Foods 2019, 8, 543
(TNF-α, IL-6, and IL-12) was suppressed by genistein below 20 μM, suggesting that genistein possesses
an immunosuppressive activity. Therefore, we selected concentrations below 20 μM of genistein
for further assessment of DC maturation. The complex process of DC maturation is accompanied
by the upregulation of the expression of MHC class II molecules and three major co-stimulatory
molecules (CD40, CD80, and CD86) on the surface of DCs. As shown in Figure 7, LPS stimulation
upregulated the expression of MHC class II and also of the co-stimulatory molecules (CD40, CD80,
and CD86) in DCs, while genistein treatment significantly decreased the expression levels of all these
molecules. These data indicated that genistein indeed impaired LPS-stimulated DC maturation at
non-cytotoxic concentrations.
Table 1. Chemical shifts (δH ) and assignment of major compounds present in the D-4 and
D-5 subfractions.
Compounds δH (mult, J in Hz)

Genistein 13.01 (s), 8.13 (s), 7.44 (d, 8.7), 6.88–6.94 1 , 6.40 (d, 2.2), 6.28 (d, 2.2)
p-Hydroxybenzoic acid 2 7.90 (d, 8.9), 6.88–6.94 1
Salicylic acid 7.88 (dd, 7.6, 1.7), 7.55 (ddd, 8.9, 7.6. 1.8), 6.88–6.94 1
Vanillic acid 7.58 (dd, 8.2, 2.0), 6.88–6.94 1 , 3.89 (s)
1 Overlapping peaks at 6.88–6.94 ppm. 2 Only in the D-4 subfraction.
Figure 6. The effect of genistein on cell viability (A) and the production of pro-inflammatory cytokines
in LPS-stimulated DCs (B, C, and D). (A) DCs were treated with genistein at various concentrations for
24 hours, and then their viability was measured by the CCK-8 assay (Sigma). DCs were untreated or
treated with LPS (100 ng/mL) and LPS + genistein (2.5, 5, 10, 20, and 40 μM), as indicated. Supernatants
were collected 6 h after treatment. The production of the cytokines TNF-α (B), IL-6 (C), and IL-12p70
(D) was measured by ELISA. The data shown are the mean ± SD of three independent experiments.
### p < 0.001; * p < 0.05; ** p < 0.01; *** p < 0.001 (Scheffe’s test) for comparisons of the genistein-treated
and untreated LPS-stimulated DCs.

Foods 2019, 8, 543
Figure 7. Genistein-mediated suppression of a maturation-associated surface marker on LPS-stimulated

DCs. DCs were untreated or treated with LPS or LPS + genistein for 24 h. The suppression of major
histocompatibility complex (MHC) class II and of co-stimulatory molecules (CD40, CD80, and CD86)
was analyzed by flow cytometry. All of the data shown were gated on CD11c+ cells. All results are
representative of three independent experiments.
Isoflavones, including genistein, daidzein, and glycitein, are generally found in leguminous plants
and have been reported as antioxidants and immunosuppressant agents, capable of suppressing the
allergic sensitization to peanuts by regulating human monocyte-derived dendritic cell function [24].
The majority of the dietary isoflavonoids are present in inactive glycosides forms (e.g., genistin) and then
converted to active aglycone forms (e.g., genistein) by the bacterial microbiote in the digestive tract. Thus,
DCs have direct access to dietary antigens and are therefore poised to uptake isoflavones directly from
the lumen [24]. Previously, genistein was shown to have promising activities, such as neuroprotective
effects by improving hippocampus neuronal cell viability and proliferation in vitro [25], antioxidant
capacity by regulating β-oxidation and energy metabolism in vivo [26], and anti-inflammatory effects by
inhibiting the ERK pathway [27] and NF-κB-dependent gene expression in TLR4-stimulated DCs [28].
In this study, the association between genistein and the immunomodulatory effect of UCME was
carefully elucidated through the combination of bioactivity-guided fractionation and NMR-based
identification. An 1 H NMR-based metabolomics approach was applied to partially purified
subfractions D-1 to D-6 from UCME. 1 H NMR profiling suggested the presence of genistein in
the D-4 and D-5 subfractions, which exhibited a stronger inhibitory activity against cytokine
production in LPS-stimulated DCs. Genistein was therefore supposed to be a possible marker
of the immunosuppressive activity of UCME. This suggestion was supported by the results of
the genistein-knockout subfractions, which showed a much lower inhibitory activity. Moreover,
HPLC isolation of the D-4 and D-5 subfractions provided other compounds with poor inhibitory
activity. On the basis of this evidence, genistein could be used as a chemical marker for the quality
control of the potentially immunosuppressive functional food UC roots. A literature survey disclosed
that the 1 H NMR-based metabolomics approach for screening bioactive secondary metabolites has not
been widely used [29,30]. Our study provides a powerful tool for discovering the active ingredients
in immunosuppressive functional foods. This approach can be applied for investigating the active
ingredients of herbal products.
Foods 2019, 8, 543
3.1. General Experimental Procedures

Column chromatography (CC) was performed on Silica gel 60 (40–63 μm, Merck, Darmstadt,
Germany). Thin-layer chromatography (TLC) was performed on silica gel 60 F254 plates (200 μm,
Merck). High-performance liquid chromatography (HPLC) was performed, using Keystone Spherisorb
silica (5 μm, 250 × 10 mm), on a Knauer Smartline 2400 refractive index (RI) detector and a Knauer
Smartline 100 pump. The NMR experiments were performed on a Bruker DRX-500 NMR spectrometer
(Bruker, Rheinstetten, Germany). Flow cytometry was conducted using a BD FACSCanto II Flow
Cytometer (BD Biosciences, CA, USA).
3.2. Sample Preparation and Isolation

UC was purchased from Mingjian Township, Nantou, Taiwan. The procedure of extraction and
isolation is summarized in Figure 2. Briefly, the roots (1.136 kg) were pulverized into a fine powder
and extracted with methanol (12 L). The supernatant was collected and concentrated under reduced
pressure to obtain the methanolic extract (UCME, 80 g). A portion of the residue (60 g) was suspended
in H2 O and sequentially fractionated with EtOAc and n-BuOH. The EtOAc-soluble fraction (9.6 g) was
then subjected to silica-gel column chromatography (150 g, 70–230 mesh), using a gradient solvent
system of n-hexane, EtOAc, and MeOH as a mobile phase. Each fraction, from which a sample was
collected for the immunomodulating assessment, was analyzed by thin-layer chromatography (TLC)
and assigned to one of 9 main fractions (Fr.A to Fr.I). A mixture of β-sitosterol and stigmasterol was
obtained from fraction C (n-hexane/EtOAc = 9/1). The subfractions of fraction D (n-hexane/EtOAc = 7/3)
were further analyzed by 1 H NMR, indicating the presence of genistein in the D-4 and D-5 subfractions.
The genistein-knockout subfractions (D-4 w/o and D-5 w/o) were obtained by HPLC and evaluated in
terms of their immunomodulating activity.
3.3. HPLC Conditions Used for the D-4 and D-5 Subfractions
D-4 and D-5 were isolated by semipreparative HPLC (dichloromethane/acetone = 85/15,
flow rate = 3 mL/min) to obtain the genistein-knockout subfractions D-4 w/o, D-5 w/o and genistein
(24.0 mg, tR = 8.0 min). The genistein-knockout subfractions D-4 w/o and D-5 w/o were further
isolated by semipreparative HPLC (n-hexane/acetone = 2/1, flowrate = 3 mL/min) to obtain salicylic
acid (tR = 8.3 min), lupinalbin A (tR = 11.0 min), 1,10-decanedioic acid (tR = 11.3 min), monomethyl
succinate (tR = 13.2 min), p-hydroxybenzoic acid (tR = 16.7 min), and vanillic acid (tR = 18.5 min).
3.4. NMR Analysis

The samples were dissolved in the deuterated solvent acetone-d6 and put into a 5 mm NMR
tube. All experiments were performed on a Bruker DRX-500 NMR spectrometer (Bruker, Rheinstetten,
Germany), operating at a frequency of 500 MHz for 1 H NMR observation, and 125 MHz for 13 C NMR
observation (at room temperature). The 2D NMR experiments included heteronuclear single-quantum
correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC). NMR spectra were carefully
processed with the TOPSPIN2.1®software (Bruker). The spectra recorded in acetone-d6 were referenced
to the solvent signal at δH 2.05 ppm and δC 29.92 ppm.
3.5. Preparation of BMDC

The ICR mice used in this study were obtained from the National Laboratory Animal Center (NLAC,
Taipei, Taiwan). The mouse bone marrow-derived DCs were prepared as described previously [31].
Bone marrow cells were isolated from tibias and femurs and then seeded on 6-well plates (Corning) in
4 mL/well RPMI 1640 medium (Thermo), with 10% FBS and 10 ng/mL recombinant mouse GM-CSF
Foods 2019, 8, 543
and IL-4 (Peprotech). On day 3 and 5, a 2 mL/well fresh medium containing 10 ng/mL GM-CSF and
IL-4 was added. On day 7, BMDCs (> 80% CD11c+ cells) were harvested and used for all experiments.
3.6. Measurement of Cytokine Production

Cytokine production was measured using an enzyme-linked immunosorbent assay (ELISA),
as described previously [31]. The DCs were treated with 100 ng/mL lipopolysaccharide (LPS) (Sigma)
or LPS + sample for 6 h for TNF-α, IL-6, and IL-12p70 determination. The production of cytokines was
measured using the ELISA kit (eBioscience).
3.7. Cytotoxicity Assessment

DCs were treated with genistein (2.5, 5, 10, 20, and 40 μM) for 24 h. The cells were then measured
in terms of their cell viability by the CCK-8 assay (Sigma), according to standard protocols, as described
previously [32]. Triplicate treatments were performed for each sample in all experiments.
3.8. Analysis of DC Maturation

Maturation was determined by measuring the upregulation of MHC class II and three
co-stimulatory molecules (CD40, CD80, and CD86), as described previously [31,32]. DCs were untreated
or treated with LPS (100 ng/mL) or LPS + genistein (5, 10, and 20 μM) for 24 h. Cell aggregation was
examined by microscopy (40×). Then, the cells were stained with monoclonal antibodies (mAbs),
specific to mouse CD11c, MHC class II, CD40, CD80, and CD86 (Biolegend), and analyzed by flow
cytometry. The fluorescence intensity of MHC class II, CD40, CD80, and CD86 was determined,
following gating with a forward side scatter (FSC) and CD11c+ expression. The change in the mean
fluorescence intensity (MFI) from LPS alone to LPS + genistein was indicated.
3.9. Data Analysis

The significance of the suppressions was determined using one-way ANOVA, followed by
Scheffe’s test. A value of * p < 0.05 was considered significant. Values of ** p < 0.01 and *** p < 0.001
were considered highly significant.
4. Conclusions
In the present study, we assessed the effect of UC roots on the immune function of DCs and found
that the immunomodulatory effect of UCME was mainly associated with its EtOAc-soluble fraction.
After one-step chromatography, genistein was rapidly identified by the 1 H NMR profiling of the
immunomodulatory subfractions (D-4 and D-5). The central role of genistein in the immunomodulatory
activity of UC roots was supported by the result that the genistein-knockout subfractions (D-4 w/o and
D-5 w/o) had a lower inhibitory activity.
Importantly, this work elucidates a rapid strategy for identifying immunomodulatory
phytochemicals, distinguishing the chemical marker(s) for quality control and providing a rationale
for the traditional immunomodulatory use of UC roots. The findings indicated that UC roots can
potentially be used as an immunosuppressive functional food, of which genistein can be a chemical
marker for quality control. In conclusion, our strategy will help in the standardization of U. crinita
roots, a famous Taiwanese functional food.

Figure S1: Selected 13 C NMR spectrum (acetone-d6 , 125 MHz) of subfraction D-4; Figure S2: Selected HSQC
spectrum (acetone-d6 ) of subfraction D-4; Figure S3: Selected HMBC spectrum (acetone-d6 ) of subfraction
D-4; Figure S4: Chromatogram of genistein-containing subfractions D-4 and D-5; Figure S5: The effects of the
compounds LA (lupinalbin A), MS (p-hydroxybenzoic acid), HA (p-hydroxybenzoic acid), DDA (p-hydroxybenzoic
acid), and ST (a mixture of β-sitosterol and stigmasterol) on the production of pro-inflammatory cytokines in
LPS-stimulated DCs; Figure S6: The effects of the compounds SA (salicylic acid) and VA (vanillic acid) on the
production of pro-inflammatory cytokines in LPS-stimulated DCs.
Foods 2019, 8, 543
Author Contributions: Conceptualization, M.-K.L. and M.-S.L.; methodology, Y.-H.K. and M.-K.L.; investigation,
P.-C.T., C.-J.C., and Y.-C.L.; resources, Y.-H.K., M.-S.L., and M.-K.L.; writing—original draft preparation, P.-C.T.
and C.-J.C.; writing—review and editing, P.-C.T., M.-K.L., and M.-S.L.; supervision, M.-K.L. and M.-S.L.
Funding: This research was funded by grants from China Medical University and the Ministry Science and
Technology of Taiwan, ROC (grant number, CMU 107-5-49 and MOST 107-2313-B-039-001).
Abbreviations
CCK-8 Cell-Counting Kit-8
CD co-stimulatory molecules
ERK extracellular-regulated protein kinases
GM-CSF granulocyte-macrophage colony-stimulating factor
HSQC heteronuclear single-quantum correlation
HMBC heteronuclear multiple-bond correlation
IL interleukin
MHC major histocompatibility complex molecules
MS mass spectrometry
NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells
NMR nuclear magnetic resonance spectroscopy
TNF-α tumor necrosis factor alpha
TLRs Toll-like receptors
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foods
Article
The Essential Oil and Hydrolats from Myristica
fragrans Seeds with Magnesium Aluminometasilicate
as Excipient: Antioxidant, Antibacterial, and
Anti-inflammatory Activity
Inga Matulyte 1,2 , Aiste Jekabsone 2 , Lina Jankauskaite 2,3 , Paulina Zavistanaviciute 4 ,
Vytaute Sakiene 4 , Elena Bartkiene 4 , Modestas Ruzauskas 5 , Dalia M. Kopustinskiene 2 ,
Antonello Santini 6, * and Jurga Bernatoniene 1,2
1 Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences,
LT-50161 Kaunas, Lithuania; inga.matulyte@lsmuni.lt (I.M.); jurga.bernatoniene@lsmuni.lt (J.B.)
2 Institute of Pharmaceutical Technologies, Medical Academy, Lithuanian University of Health Sciences,
LT-50161 Kaunas, Lithuania; aiste.jekabsone@lsmuni.lt (A.J.); lina.jankauskaite@lsmuni.lt (L.J.);
daliamarija.kopustinskiene@lsmuni.lt (D.M.K.)
3 Department of Pediatrics, Lithuanian University of Health Sciences Hospital Kauno Klinikos,
LT-50161 Kaunas, Lithuania
4 Department of Food Safety and Quality, Lithuanian University of Health Sciences, LT-47181 Kaunas,
Lithuania; paulina.zavistanaviciute@lsmuni.lt (P.Z.); vytaute.sakiene@lsmuni.lt (V.S.);
elena.bartkiene@lsmuni.lt (E.B.)
5 Institute of Microbiology and Virology, Lithuanian University of Health Sciences, LT-47181 Kaunas,
Lithuania; modestas.ruzauskas@lsmuni.lt
6 Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy
* Correspondence: asantini@unina.it
Received: 10 December 2019; Accepted: 30 December 2019; Published: 2 January 2020
Abstract: Nutmeg (Myristica fragrans) essential oil has antimicrobial, antiseptic, antiparasitic,
anti-inflammatory, and antioxidant properties. We have recently demonstrated that hydrodistillation
of nutmeg essential oil by applying magnesium aluminometasilicate as an excipient significantly
increases both the content and amount of bioactive substances in the oil and hydrolats. In this
study, we aimed to compare the antioxidant, antimicrobial, and anti-inflammatory activity of
hydrolats and essential oil obtained by hydrodistillation in the presence and absence of magnesium
aluminometasilicate as an excipient. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
method revealed that magnesium aluminometasilicate did not significantly improved antioxidant
activity of both essential oil and hydrolat. Antibacterial efficiency was evaluated by monitoring growth
of 15 bacterial strains treated by a range of dilutions of the essential oil and the hydrolats. Essential oil
with an excipient completely inhibited the growth of E. faecalis, S. mutans (referent), and P. multocida,
whereas the pure oil was only efficient against the latter strain. Finally, the anti-inflammatory
properties of the substances were assessed in a fibroblast cell culture treated with viral dsRNR mimetic
Poly I:C. The essential oil with an excipient protected cells against Poly I:C-induced necrosis more
efficiently compared to pure essential oil. Also, both the oil and the hydrolats with aluminometasilicate
were more efficient in preventing IL-6 release in the presence of Poly I:C. Our results show that the
use of magnesium aluminometasilicate as an excipient might change and in some cases improve the
biological activities of nutmeg essential oil and hydrolats.
Keywords: nutmeg; essential oil; antioxidant activity; antibacterial activity; poly I:C-induced
inflammation; fibroblasts; magnesium aluminometasilicate

Foods 2020, 9, 37
1. Introduction
Since ancient times, Myristica fragrans (nutmeg) seeds have been used as a food spice, flavoring
agent, a natural remedy for headaches and fever [1]. Nutmeg seeds have essential and fatty oils, resins,
wax, and other components [2]. Nutmeg’s essential oil has antimicrobial, antiseptic, antiparasitic,
anti-inflammatory, and antioxidant properties [1,3]. The concentration of essential oil in nutmeg seeds
is about 5–15% [4], and its major components are terpene hydrocarbons (sabinene, pinene, camphene,
p-cymene, phellandrene, terpinene, limonene, and myrcene altogether make up 60% to 80% of the oil),
oxygenated terpenes (linalool, geraniol, and terpineol, which make up approximately 5% to 15%) and
aromatic ethers (myristicin, elemicin, safrole, eugenol, and eugenol derivatives, together constituting
15 to 20%) [5–8]. The toxicity of nutmeg seeds at high doses has been reported, mainly due to myristicin
oil and elemicin, causing tachycardia, nausea, vomiting, agitation, and hallucinations. However, these
effects are related to the abuse of the spice and are not observed at usual low concentrations [9]. There
are many studies on the beneficial effects of nutmeg seed and various nutmeg seed extracts. One of the
most prominent biological activities of the nutmeg preparations is antibacterial. Nutmeg seed lignans
exert antimicrobial activity on Bacillus subtilis, Staphylococcus aureus, and Shigella dysenteriae [10]. Ethanol
and acetone extracts of nutmeg crust have strong antibacterial activity against gram-positive bacteria
Staphylococcus aureus [5]. Ethyl acetate extracts of flesh of the nutmeg fruit have inhibitory potential
against both gram-positive and gram-negative bacteria with a minimum inhibitory concentration (MIC)
ranging from 0.625 to 1.25 mg/mL [11]. Used for the preservation of sweets, nutmeg methanol extracts
inhibit growth of Staphylococcus aureus, Aspergillus niger, Saccharomyces cerevisiae, and Escherichia coli
at MIC between 250 and 300 mg/mL [12]. However, there are only a few studies on the biological
activity of nutmeg essential oil. Takikawa et al. showed a higher antibacterial effect of essential nutmeg
oil on pathogenic compared to non-pathogenic strains of Escherichia coli [13]. Furthermore, nutmeg
essential oil decreased the growth and survival of Yersinia enterocolitica and Listeria monocytogenes in
broth culture [14].
Nutmeg oil preparations are also known for their antioxidant capacity. Using the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) free radical scavenging assay, Piaru et al. reported a significant antioxidant
activity of nutmeg oil [15]. The antioxidant properties are often related to the alleviation of inflammation.
Nutmeg oil diminished chronic inflammation and pain through the inhibition of COX-2 expression
and substance P release in vivo [16]. In another study, nutmeg oil suppressed reactive oxygen species
(ROS) production in human neutrophils stimulated by PMA (phorbol 12-myristate 13-acetate) [17] and
mildly inhibited phagocytosis in human neutrophils [18]. However, there is no published research on
the effect of nutmeg seed essential oil on virus-triggered inflammatory response.
Hydrodistillation is a popular method used for the preparation of essential oils. However,
hydrodistillation with excipients is not widely used—we have found just three studies applying this
method so far [19–21]. Therefore, we have applied magnesium aluminometasilicate in hydrodistillation
as the new excipient and have tested its effects on the nutmeg essential oil yield and its composition [22].
Aluminometasilicate is widely used as a disintegrator in the manufacturing of tablets. Furthermore,
this compound is non-toxic and inexpensive, as the price is ~300 eur for 25 kg. Magnesium
aluminometasilicate has significantly increased both the yield and composition of some chemical
compounds (sabinene, α-pinene, and limonene). The use of the excipient also increased the essential oil
yield by about 61% (hydrodistillation with water—the yield is 0.79 ± 0.04 g, using 1% excipient—1.29
± 0.05 g; the nutmeg quantity was 15 g, the water content was 300 mL) [22].
The increased amount of active substances suggests that oil preparations with aluminometasilicate
might have stronger biological activities. Therefore, in this study we compared the antioxidant,
antimicrobial, and anti-inflammatory properties of Myristica fragrans seed essential oil preparations
with and without aluminometasilicate.
Foods 2020, 9, 37
2.1. Plant Material

The dried seeds of nutmeg (Myristica fragrans) were from Grenada. Seeds were identified by
Jurga Bernatoniene, Medical Academy, Lithuania University of Health Sciences, Kaunas, Lithuania.
A voucher specimen (I 18922) was placed for storage at the Herbarium of the Department of Drug
Technology and Social Pharmacy. The seeds had a characteristic odor, a strong, bitter, and spicy flavour,
and they were a brown-beige color. The seeds were ground into a powder (using laboratory mill), with
particles smaller than 0.5 mm. All powder samples were kept in a dark and airtight container at 20 ±
2 ◦ C.
2.2. Essential Oil and Hydrolat

The essential oil from nutmeg seeds was prepared by using hydrodistillation. The modified
Clevenger type apparatus was used. Two samples of essential oil were prepared: one without excipient
and the other with 1% of magnesium aluminometasilicate. Each sample was prepared with 15 g of
nutmeg powder and 300 mL distilled water, and 1% magnesium aluminometasilicate was used as an
excipient in one of the samples. Also, hydrolat of these two essential oils was used. It was collected
from Clevenger apparatus. This material was collected first, followed by the essential oil. All samples
were obtained and stored in airtight bottles in the refrigerator. The hydrodistillation took 4 h.
2.3. Antioxidant Activity by DPPH Radical Scavenging Assay

Antioxidant activity of nutmeg essential oil and hydrolat were evaluated using DPPH
(Sigma Aldrich, St. Louis, MO, USA) [15]. First of all, 0.1 mM 96% DPPH solution in 96% ethanol
was prepared. A total of 1 mL of DPPH solution was placed in a spectrophotometer cuvette and 100
μL of ethanolic essential oil solution at concentrations ranging from 0.2% to 20% was added. For an
antioxidant activity evaluation of hydrolat, the absolute hydrolat was used. 1 mL DPPH solution and
nutmeg hydrolat from 0.1 mL to 1 mL were mixed in a cuvette. All samples were incubated in the
dark for 20 min and absorbance was taken at 515 nm. The antioxidant activity was performed on a
UV Spectrophotometer UV-1800 (Shimadzu, Kyoto, Japan). The quantity of DPPH radical scavenging
activity was calculated by using this formula:
Acontrol − Asample
DPPH scavenging e f f ect % = × 100, (1)
Acontrol
where Acontrol and Asample are the absorbance of the control sample (0.1 mM DPPH solution, solvent is
96% ethanol) and the experiment sample.
2.4. Antimicrobial Activity

The method used for antimicrobial activity was serial dilutions in liquid medium [23]. The broth
liquid medium was dispensed into test tubes to give a final volume of 10 mL (with a sample of essential
oil). The medium was sterilized. The physiological solution was dispensed into 5 mL individual tubes
and used for preparation of suspension of the following bacteria: Klebsiella pneumoniae; Salmonella enterica
24 SPn06; Pseudomonas aeruginosa 17–331; Acinetobacter baumanni 17–380, Proteus mirabilis; 6MRSA
M87fox; Enterococcus faecalis 86; Enterococcus faecium 103; Bacillus cereus 18 01; Streptococcus mutans
(referent); Enterobacter cloacae; Citrobacter freundii; Staphylococcus epidermidis; Staphylococcus haemolyticus;
Pasteurella multocida strains. All bacteria were isolated from clinical material. For each bacterial culture,
three tubes of Mueller Hinton broth were used (9.94 mL, 9.97 mL, and 9.98 mL each). The tubes were
inoculated with 10 μL of bacterial suspension with the essential oil at concentration 0.1%, 0.2%, and
0.5%. After 48 h of incubation, each tube was inoculated with 10 μL of suspension on soy-tryptone
agar (Thermo Fisher, Hampshire, UK). MIC of essential oil was evaluated based on the presence of
bacterial growth (bacterial colonies growing (+)/non growing (−).
Foods 2020, 9, 37
2.5. Cell Culture and Treatments

Human fibroblasts (BJ-5ta, hTERT, LGC Standards Ltd. Middlesex, UK) were grown in 75 cm2
flasks in Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax (Thermo Fisher Scientific,
Waltham, MA, USA), 10% fetal bovine serum and 100 IU/mL Penicilin/Streptomycin according to
standard supplier protocol. At 70–90% confluence, the cells were detached by 0.025% Trypsin/EDTA
and plated in 96 well plates at a density of 2 × 105 cells/well. A total of 24 h after plating, the cells
were treated with 1 μg/mL Poly I:C to simulate viral dsRNR-induced inflammatory response. For cell
culture treatments, the essential oils were dissolved in 96% ethanol at a concentration of 5% (v/v).
For determination of cell viability and LD50 , the solutions of essential oils and absolute hydrolats
were used as a range of dilutions in cell culture medium starting from essential oil preparation to a
medium v/v ratio of 1:1000 and finishing with 1:5. For the control, the same dilutions with solvent
(ethanol) were performed. For anti-inflammatory activity evaluation, the solutions of essential oils
at v/v dilutions of 1:100 or 1:200, or absolute hydrolats at v/v dilutions of 1:40, 1:100, and 1:200 were
applied simultaneously with Poly I:C treatment.
2.6. Determination of Cell Viability and Determination on LD50

Cell viability was assessed by using double nuclear fluorescent staining with Hoechst 33342
(10 μg/mL) and propidium iodide (PI, 5 μg/mL) according to standard supplier protocol for 5 min at
37 ◦ C. PI-positive nuclei indicating lost nuclear membrane integrity were considered to be necrotic.
Cells were visualized under fluorescent microscope OLYMPUS IX71S1F-3, counted in fluorescent
micrographs and expressed as percentage of total cell number per image. The data is presented as
averages ± standard deviation. LD50 was calculated by SigmaPlot v.13 (Systat Software Inc., San Jose,
CA, USA) using the equation selected by a dynamic curve fitting tool.
2.7. Assessment of Interleukin-6 Concentration

Medium collected after cell culture treatments was used to measure the concentration of
pro-inflammatory cytokine interleukin-6 (IL-6) by ELISA kit (Thermo Fisher Scientific, Waltham,
MA, USA) following the standard supplier protocol. The spectrophotometric readings were performed
in a plate reader Infinite 200 Pro M Nano Plex (Tecan, Mannedorf, Svizzera).

The results are presented as means of 3–7 replicates ± standard deviation. The statistical data
analysis was performed by applying ANOVA with Tukey HSD post hoc test. Differences were
considered statistically significant when p < 0.05. The data were processed using Microsoft Office Excel
2010 (Microsoft, Redmond, WA, USA) software.
3. Results
First, the antioxidant activity in essential oils was compared. The two samples of each category
were analyzed: the essential oil without excipient, or pure essential oil (EO1), and essential oil with 1%
of magnesium aluminometasilicate (EO2). The results are presented in Table 1. The range of essential
oil concentration in this examination was from 0.2 to 20%.
Table 1. Antioxidant activity of nutmeg essential oils applied at different concentrations.
Essential Oil Concentration (%)

Sample
0.2 0.5 1 2 5 10 20
EO1 12.63 ± 0.53 16.34 ± 1.23 26.35 ± 0.88 30.58 ± 1.39 44.53 ± 0.84 61.01 ± 0.26 84.01 ± 0.78
EO2 12.65 ± 2.05 19.12 ± 2.24 27.03 ± 0.98 37.15 ± 0.80 * 44.92 ± 0.63 62.11 ± 0.43 72.71 ± 0.79 *
*—significant difference compared to EO1, p < 0.05, n = 3.
Foods 2020, 9, 37
Both essential oils demonstrated similar antioxidant activity increasing in a concentration-

dependent manner, except for some small fluctuations at 2% (EO2 had slightly higher antioxidant
activity compared to EO1) and 20% (EO2 antioxidant activity was slightly lower than EO1). Both
essential oil preparations at 10% concentration had higher than 50% antioxidant activity (more than
half of DPPH radicals were bound).
Next in the study, the antioxidant activity of essential oil hydrolats was tested by using DPPH
radical scavenging method. Hydrolat from EO1 was named EOH1, and from hydrolat from EO2 was
named EOH2. The data are provided in Table 2.
Table 2. Antioxidant activity of nutmeg essential oil hydrolats.
Hydrolat Quantity (mL)

Sample
0.1 0.2 0.3 0.5 1
EOH1 12.97 ± 1.25 31.43 ± 1.55 36.21 ± 3.20 48.09 ± 3.96 56.42 ± 3.23
EOH2 15.22 ± 5.14 27.24 ± 1.63 33.52 ± 2.11 36.55 ± 0.68 * 44.19 ± 1.09 *
*—significant difference compared to EOH1, p < 0.05, n = 3.
At small quantities of up to 0.3 mL, the antioxidant activities of both hydrolat preparations were
similar, but at 0.5 mL and 1 mL, the EOH1 antioxidant activity was significantly higher compared to
that of EOH2. EOH1 at 1 mL had an antioxidant activity greater than 50%, and this activity level was
similar to 5%–10% of EO1 activity. The results show that free radical scavenging activity of 0.2 mL of
hydrolat is higher than that of 1% or less concentrated essential oil.
Summarizing the antioxidant activity results, magnesium aluminometasilicate did not improve,
and even slightly decreased the antioxidant activity of nutmeg essential oil and its hydrolat.
Next in the study, antibacterial properties of nutmeg essential oil and hydrolats were investigated
on 15 pathogenic clinical isolate strains by using a dilution range assay. The results are presented in
Table 3.
Table 3. Antimicrobial study results of nutmeg essential oil and its hydrolats.
Microorganisms
Sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
EO1 + + + + + + + + + + + + + + 0.2
EO2 + + + + + + 0.5 + + ≤0.1 + + + + 0.2
EOH1 + + + + + + + + + + + + + + +
EOH2 + + + + + + + + + 0.5 + + + + +
+ means the pathogens growth. 1. Klebsiella pneumoniae, 2. Salmonella enterica 24 SPn06, 3. Pseudomonas
aeruginosa 17-331, 4. Acinetobacter baumanni 17-380, 5. Proteus mirabilis, 6. 6MRSA M87fox, 7. Enterococcus
faecalis 86, 8. Enterococcus faecium 103, 9. Bacillus cereus 18 01, 10. Streptococcus mutans (referent), 11. Enterobacter
cloacae, 12. Citrobacter freundii, 13. Staphylococcus epidermidis, 14. Staphylococcus haemolyticus, 15. Pasteurella
multocida. Where the growth of bacteria were inhibited, minimal inhibitory concentrations were provided in %.
EO1 only suppressed Pasteurella multocida growth, with the minimal concentration to achieve
this effect being 0.2%. However, nutmeg essential oil with 1% of magnesium aluminometasilicate
(EO2) had a broader effect. Next to P. multocida, it inhibited E. faecalis and S. mutans, and the efficient
concentrations were rather low. A mere 0.5% was enough to completely suppress E. faecalis, and
for the S. mutans strain even less than 0.1% was effective. In the case of EOH1 and EOH2, only the
hydrolat with aluminometasilicate suppressed the growth of S. mutans. Thus, the results indicate
that the excipient magnesium aluminometasilicate broadens the spectrum of antimicrobial activity of
nutmeg essential oil.
One of the most important pharmacological activities of plant essential oils is related to
anti-inflammatory properties. Nutmeg essential oil is also known for inflammation reducing
activity [24]. Therefore, next in the study, we have assessed nutmeg seed essential oil and hydrolat
Foods 2020, 9, 37
preparations in a virus mimetic Poly I:C-induced inflammation in vitro model by using human
fibroblast cell culture. Before starting the treatments, the general toxicity test of the oils and hydrolats
was performed and LD50 doses as well as safe concentrations were established.
As indicated in Figure 1a, there were no significant difference in cell viability detected after
treatment with both EO1 and EO2 essential oil solutions up to the dilution ratio 1:100. Further increases
in concentration up to the dilution ratio 1:40 significantly decreased viability of the fibroblasts. After
treatment with essential oil solutions at 1:40, the viability dropped from 97 ± 2% in control to 70 ± 12%
in the case of EO1, and to 42 ± 10% in the case of EO2. At the dilution ratio of 1:5, the percentage of
viable cells in the cultures was lower than 10% in the case of both essential oil preparations. The dilution
ratios corresponding to LD50 calculated for EO1, EO2, and 96% ethanol were 0.047, 0.022, and 0.055,
respectively. Thus, EO2 was significantly more toxic for the cells compared to EO1, and also to ethanol.
In contrast, the toxicity pattern of EO1 was very close to that of ethanol, indicating there were no or
very little toxic compounds in this essential oil preparation.
Figure 1. The effect of nutmeg essential oil ethanol solutions (a) and nutmeg essential oil hydrolats (b)
on viability of cultured human fibroblasts. EO1—essential oil without excipient solution, EO2—essential
oil with 1% magnesium aluminometasilicate solution, EOH1—hydrolat from EO1, and EOH2—hydrolat
from EO2. In addition, 96% ethanol was assessed as solvent control for the essential oil. Punctured
lines indicate the dilution ratios corresponding to LD50 . *—statistically significant difference compared
to untreated control, #—compared to EO1 in (a) or EOH1 in (b), respectively, when p < 0.05.
Evaluation of cell viability after 24 h treatment with nutmeg seed essential oil hydrolats revealed
that both EOH1 and EOH2 were not toxic up to a dilution of 1:20 (Figure 1b). After cell incubation with
1:10 EOH2, the percentage of viable cells in the cultures decreased to 57 ± 19%, making a significant
difference compared with the untreated control. A significant viability drop in EOH1 treatment series
was achieved when the dilution ratio 1:5 was applied. The level of viable cells in this treated cultures
was 44 ± 14%. After treatment with EOH1 and EOH2 at the ratio 1:2, nearly all cells in the cultures
were found to be necrotic. The dilution ratios corresponding to LD50 calculated for EOH1 and EOH2
were 0.160 and 0.105, respectively. Toxicity evaluation of the hydrolats indicated that EOH2 is slightly
more toxic compared to EOH1.
The next task in this work was to evaluate the efficiency of nutmeg seed essential oil and hydrolat
preparations to reduce toxicity and signaling in viral inflammation in vitro model. To stimulate
inflammatory response, human fibroblast cell culture was treated with 1 μg/mL virus double stranded
RNR mimetic polyinosinic: polycytidylic acid (Poly I:C) for 24 h, with or without nutmeg seed essential
oil solutions or hydrolats. After toxicity assessment, the dilution ratios selected for anti-inflammatory
property testing were 1:200 and 1:100 for the essential oil solutions, and 1:100 and 1:40 for the hydrolats.
Anti-inflammatory assessment results are presented in Figure 2.
Foods 2020, 9, 37
Figure 2. The effect of nutmeg seed essential oil ethanol solutions and the essential oil hydrolats on Poly
I:C-treated human fibroblast cell viability (a) and cytokine IL-6 release from the cells (b). EO1—essential
oil without the excipient solution, EO2—essential oil with 1% magnesium aluminometasilicate solution,
EOH1—hydrolat from EO1, and EOH2—hydrolat from EO2. *—statistically significant difference
compared to untreated control, #—compared to Poly I:C-only treatment, when p < 0.05; n = 5–7.
After fibroblast cell culture treatment with 1 mg/mL Poly I:C, the amount of viable cells decreased
by 59% (Figure 1a). Addition of nutmeg essential oil preparations to the cell culture medium increased
cell viability in the Poly I:C-affected cultures. Statistically significant differences compared with Poly
I:C samples were found after treatment with 1:200 EO1, 1:200 and 1:100 EO2, as well as 1:40 EOH1 and
1:40 EOH2. The percentages of viable nuclei in these samples were 81 ± 9%, 82 ± 16%, 76 ± 10%, 72 ±
14%, and 79 ± 12%, respectively. Thus, EO2 has demonstrated the highest cytoprotective capacity in a
virus mimetic inflammation model.
Evaluation of the release of IL-6 to the incubation medium revealed that after 24 h of Poly I:C
treatment, the level of this pro-inflammatory cytokine jumped from nearly a “zero” value to 883 ±
273 pg/mL (Figure 2b). Nutmeg essential oil preparations applied together with Poly I:C significantly
reduced the concentration of IL-6 in the medium. The significant drop in the IL-6 level was in the
samples incubated with 1:100 EO1, 1:200 and 1:100 EO2, 1:40 EOH1, and 1:100 and 1:40 EOH2. IL-6
concentration in these samples was found in the range between 162 ± 123 pg/mL (with 1:40 EOH2) and
206 ± 83 pg/mL (with 1:100 EO1). The assessment of IL-6 release indicates that both the solution of
nutmeg seed essential oil with magnesium aluminometasilicate and the hydrolat from this essential oil
are most efficient against Poly I:C-induced release of this inflammatory cytokine.
4. Discussion
The purpose of our study was to compare the biological activity of nutmeg seed essential oil
and hydrolat without excipient and using magnesium aluminometasilicate as the excipient. To our
knowledge, it is the first application of aluminometasilicate as an excipient in essential oil studies.
Essential oils have strong antioxidant activity, and some of them are used as preservation agents
protecting food or cosmetics from oxidation-induced spoilage [25,26]. Antioxidant activity studies
help to elucidate essential oil capacity to protect food from free radical damage [27]. The DPPH radical
scavenging method is widely used for this purpose because it is simple and cost-efficient, and gives
reliable results. Therefore, it was selected to evaluate the essential oil and hydrolat preparations in our
study. Our previous study [22] showed that magnesium aluminometasilicate had influence not only
on the yield of essential oil, but also on its chemical composition. Magnesium aluminometasilicate
significantly increased the quantity of sabinene, α-pinene, and limonene. Dai et al. (2013) study with
Wedella Prostrata essential oil (containing 11.38% limonene and 10.74% α-pinene) had a lower antioxidant
activity than 100 μg/mL limonene but a higher antioxidant activity than the pure α-pinene [28]. Such a
result suggests that limonene is a more prominent antioxidant compared to α-pinene. Our nutmeg seed
essential oil (EO2) had 11.66 ± 3.39% α-pinene and 4.91 ± 0.71% limonene [22]. Based on the results
of the study where the presence of limonenen together with α-pinene resulted in higher antioxidant
activity [28], we can predict that our EO2 has higher antioxidant activity than the pure α-pinene sample.
Foods 2020, 9, 37
The Juniperus scopulorum 10% essential oil had a 54.7% antioxidant activity (composition: sabinene
50.7%, α-pinene 3.23%, limonene 2.22%, cis sabinene hydrate 0.58%) [29]. Our study shows that 10%
essential oils EO1 and EO2 have 61.01 ± 0.26% and 62.11 ± 0.43% antioxidant activity, respectively.
Predominant compounds in EO2 were sabinene 61.42%, cis sabinene hydrate 0.3%, limonene 5.62%, and
α-pinene 15.05%. The EO1 essential oil had more β-pinene [22], which could increase its antioxidant
activity. Other authors have demonstrated nutmeg essential oil with higher antioxidant activity besides
α-pinene had also β-pinene [27]. Misharina et al. (2009) have also studied antioxidant properties of
nutmeg essential oil and found that 16.5% concentrated solution had approximately 50% antioxidant
activity [30]. Such differences may be due to the distinct technique of the research and the variation in
nutmeg seed material.
Hydrolats analyzed in our study had lower antioxidant activity compared to essential oils, most
likely because of the lower concentrations of volatile compounds. We have searched the literature data,
but could not find any studies about the antioxidant activity of nutmeg seed hydrolats so far. Hydrolats
prepared from other plant sources had different antioxidant properties. For example, Salvia officinalis
0.1 g/mL had about 30% radical scavenging activity. The same concentration of Rosmarinus officinalis
hydrolat had about 50% activity [31]. Our hydrolats (0.1 g/mL) had 31.43 ± 1.55% (EO1) and 27.24
± 1.63% (EO2) antioxidant activity—the same as Salvia officinalis. Nutmeg seeds hydrolats at the
highest concentration tested (0.5 g/mL) had 56.42% and 44.19% antioxidant activity (EOH1 and EOH2,
respectively).
Essential oils are known for bioactive compounds with antibacterial activity, therefore they are
used as antimicrobial agents in medicine, pharmacy, cosmetology, and other fields [18]. However,
different essential oils affect microorganisms in distinct ways—some suppress gram-positive effects,
others suppress gram-negative effects [19]. Also, the effective concentration of essential oils vary.
There are various methods for determining antibacterial activity (the agar disk-diffusion method,
antimicrobial gradient method, dilution methods, and other methods) [32]. Dilution methods are
the simplest methods used to determine whether the essential oil suppresses the growth of bacteria
or not [23]. There are many techniques and methods used for antimicrobial activity evaluation,
and therefore, it is difficult to compare the results obtained from the different studies. In our study,
the EO1 essential oil (0.2%) only suppressed Pasteurella multocida. The essential oil EO2 with a higher
quantity of sabinene, α-pinene, and limonene [22] had antimicrobial activity against three pathogens.
Next to P. multocida, it also prevented growth E. faecalis of and S. mutans. The increased efficiency
of EO2 against pathogenic strains can be explained by the higher quantity of volatile compounds.
Nurjanah et al.’s (2017) study showed (an in vitro disc diffusion antimicrobial activity method) that
Myristica fragrans essential oil (60% concentration was used) from Central Java inhibited the largest
areas [33]. The inhibition areas were from 12.96 mm to 16.79 mm, with the control at 0 mm (S. aureus,
S. dysenteriae, S. typhi, and S. epidermidis). In the essential oil used for the above-mentioned study,
sabinene, α-pinene, and β-pinene quantities were the highest out of all of the chemical compounds
(the concentrations were 18.82%, 16.54%, and 13.82, respectively). The essential oil EO2 investigated
in this study has a similar composition, meaning it could also be efficient against these pathogens
at higher concentrations. In another study, the nutmeg essential oil with similar quantity of volatile
compounds had a significant effect on the inhibition of the growth of E. coli and S. aureus [34]. In this
study, we found that essential oil EA2 (0.5%) inhibited E. faecalis. This bacteria resides in infected
canals of teeth and is often found in the oral cavity after tooth canal repair [35]. Repeated oral care
products with chlorhexidine promotes the development of E. faecalis resistance [36]. Since EA2 showed
activity against E. faecalis, nutmeg essential oil could be recommended as a safe protective component
for oral care products in the future.
The investigation of human fibroblast cell culture affected by virus mimetic Poly I:C showed
that nutmeg essential oils and hydrolats have an anti-inflammatory effect protecting cell viability
and significantly reducing the release of cytokine IL-6. EO2 had a higher effect on preventing Poly
I:C-induced necrosis and both EO2 and EOH2 more efficiently protected against IL-6 release compared
Foods 2020, 9, 37
to preparations without aluminometasilicate EO1 and EOH1. This is most likely due to the increased
amount and content of active substances (sabinene, α-pinene, and limonene) in the preparations that is
a result of the use of the excipient. α-Pinene significantly decreases the LPS-induced production of IL-6,
TNF-α and nitric oxide in bacterial lipopolysaccharide (LPS)-treated macrophages [37]. Sabinene from
Oenanthe crocata essential oil significantly inhibits nitric oxide production in LPS and IFNγ-treated
macrophages [38]. Limonene has a significantly decreased manifestation of inflammatory signals
in rat models of ulcerative colitis via regulation of iNOS, cyclooxygenase-2 (COX-2), PGE2, and
ERK [39]. However, there are not many studies about the anti-inflammatory effect of nutmeg essential
oil preparations. Zhang et al. have demonstrated the anti-inflammatory activity of nutmeg oil in
complete Freund’s adjuvant-injected rats [16]. Their study shows that nutmeg oil is effective in
inflammatory pain relief via inhibition of the COX-2 pathway and substance P release. Another in vivo
study exploring carrageenan-induced paw edema in rats have also confirmed the anti-inflammatory
properties of nutmeg oil [24]. To the best of our knowledge, there are no in vitro studies on virus-induced
anti-inflammatory activity of nutmeg oil. Dewi et al. have found that M. fragrans seed ethanolic extract
and pure quercetin extract from M. fragrans inhibited NO production and the release of inflammatory
cytokines, such as TNF-α, IL-6, and IL-1β from bacterial LPS-stimulated murine macrophages (RAW
264.7) in a dose-dependent manner. The essential oil of Monodora myristica was found to inhibit
inflammation-related lipoxygenase [40]. Because of the high content of bioactive volatile compounds,
which have been widely studied and characterized by gas chromatographic techniques [41] the
essential oils might be good candidates for inhalation treatment of respiratory tract infections. Most
common respiratory infections are induced by respiratory viruses, such as influenza or respiratory
syncytial virus [42]. As a result, we examined the anti-inflammatory efficiency of nutmeg essential oil
preparations in a virus mimetic Poly I:C mediated inflammation. Fibroblasts are multifunctional cells
that are responsible for support of other, more tissue-specific cell types, regeneration, wound healing,
extracellular matrix production, and inflammatory response [43]. They significantly contribute to the
response to infection by secreting cytokines for monocyte/macrophage attraction and their conversion
to inflammatory phenotype [44]. The release of IL-6 is one of the key inflammatory signals causing
activation of matrix metalloproteinases, macrophages, neutrophil production, and is also involved in
autoimmune responses in the condition such as chronic arthritis, osteoporosis, and psoriasis [41,45–48].
The results of our study indicate that nutmeg essential oil preparations have anti-inflammatory
properties that might be exploited further for treatment or prevention of viral inflammation-related
pathologies, taking the recent emerging nanotechnological and nutraceutical approaches in the field
into account [49–51]. However, to increase the applicability of these substances, more studies have to be
performed analyzing the mechanism of action of the essential compounds contained in the preparations.
5. Conclusions
Nutmeg essential oil prepared with and without magnesium aluminometasilicate as an excipient
has similar antioxidant activity. Nutmeg essential oil hydrolat prepared without excipient has a higher
antioxidant activity compared to that with magnesium aluminometasilicate as an excipient.
Nutmeg essential oil with aluminometasilicate has extended antibacterial properties compared to
the pure oil without additions. Both preparations prevent growth of P. multocida strain, but the oil with
aluminometasilicate also inhibits E. faecalis and S. mutans (referent).
Nutmeg essential oil preparations with aluminometasilicate have stronger anti-inflammatory
activity in Poly I:C-affected fibrolast cell culture. The oil with the excipient has a higher degree of
cytoprotection from Poly I:C-induced necrosis, and both the oil and hydrolats with excipient more
efficiently prevent IL-6 release compared to the preparations without aluminometasilicate.
The results show that the application of magnesium aluminometasilicate as an excipient in
hydrodistillation could help to increase the biological activity of essential oil and hydrolats.
Foods 2020, 9, 37
Author Contributions: Conceptualization, I.M. and J.B.; methodology, I.M., A.J., D.M.K., M.R., A.S. and
E.B.; validation, V.S., P.Z., J.B., A.S. and A.J.; investigation, I.M., L.J., P.Z., M.R. and V.S.; data curation, J.B.;
writing-original draft preparation, I.M., A.J., P.Z., D.M.K.; writing-review and editing, J.B. and I.M.; visualization,
L.J., I.M., G.L., A.S., V.S.; supervision, J.B. All authors have read and agreed to the published version of
the manuscript.
Acknowledgments: The authors would like to thank Open Access Centre for the Advanced Pharmaceutical
and Health Technologies (Lithuanian University of Health Sciences) and for the opportunity to use modern
infrastructure and perform this research.
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foods
Article
Elderberry (Sambucus nigra L.) Fruit Extract Alleviates
Oxidative Stress, Insulin Resistance, and
Inflammation in Hypertrophied 3T3-L1 Adipocytes
and Activated RAW 264.7 Macrophages
Joanna Zielińska-Wasielica 1 , Anna Olejnik 1, *, Katarzyna Kowalska 1 , Mariola Olkowicz 2 and
Radosław Dembczyński 1
1 Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences,
Wojska Polskiego 48, 60-627 Poznan, Poland
2 Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo,
ON N2L 3G1, Canada
* Correspondence: anna.olejnik@up.poznan.pl; Tel.: +48-61-846-60-08
Received: 11 July 2019; Accepted: 4 August 2019; Published: 8 August 2019
Abstract: Oxidative stress and inflammation in hypertrophied adipose tissue with excessive fat
accumulation play a crucial role in the development of obesity and accompanying metabolic
dysfunctions. This study demonstrated the capacity of elderberry fruit (EDB) extract to decrease the
elevated production of reactive oxygen species in hypertrophied 3T3-L1 adipocytes. Treatment with
the EDB extract resulted in modulation of mRNA expression and protein secretion of key adipokines
in hypertrophied adipocytes. Expression of leptin and adiponectin was, respectively, down- and
up-regulated. Moreover, glucose uptake stimulation was noticed in mature adipocytes, both sensitive
to insulin and insulin resistant. This may suggest a positive effect of EDB extract on insulin resistance
status. The extract was also found to alleviate the inflammatory response in activated RAW 264.7
macrophages by down-regulating the expression of proinflammatory genes (TNF-α, IL-6, COX-2,
iNOS) and suppressing the enhanced production of inflammatory mediators (TNF-α, IL-6, PGE2 ,
NO). In vitro experiments showed that the EDB extract could inhibit digestive enzymes, including
α-amylase, α-glucosidase, and pancreatic lipase, leading to reduced intestinal absorption of dietary
lipids and carbohydrates. Further in vivo studies could be postulated to support EDB as a functional
food component for the prevention and treatment of obesity and metabolic-immune comorbidities.
Keywords: elderberry polyphenols; functional food; obesity; digestive enzymes; fat cells; intracellular
reactive oxygen species; adipokines; glucose uptake; immune-metabolic effects
1. Introduction
Obesity is associated with excessive adipose tissue growth, which occurs through two possible
mechanisms: hypertrophy (expansion of existing adipocytes) and hyperplasia (recruitment of new
adipocytes). Hypertrophic adipose tissue growth is mainly considered to be related to insulin resistance
and other obesity metabolic comorbidities [1]. Abnormal expansion of adipose tissue is accompanied
by local hypoxia, adipocyte death, enhanced cytokine and chemokine secretion, dysfunctional fatty
acid metabolism and accumulation, and immune cell infiltration. Dysregulation of lipid metabolism in
adipose tissue leads to enhanced release of free fatty acids, which initiates inflammatory signaling
cascades in the infiltrating cell population. Chronic low-grade inflammation, found in abnormal fat
tissue, negatively affects the insulin signal transduction pathway, and promotes insulin resistance [2,3].
Recent scientific preclinical studies have shown that bioactive dietary compounds may specifically
influence hypertrophic adipose cells and mitigate the effects of extensive adipose tissue growth

Foods 2019, 8, 326
by affecting various adverse phenomena, including oxidative stress, inflammation, disturbances in

adipokine secretion, fatty acid release, and others. Berry fruits have been recognized as capable of
counteracting obesity and obesity-related metabolic disorders, through the inhibition of adipocyte
differentiation, a decrease in lipogenesis, an increase in lipolysis, or mitigation of inflammatory and
insulin resistance status [4].
A promising candidate capable of attenuating obesity and complications related to excessive
fat tissue growth might be Sambucus nigra L. (European elderberry) fruit as a valuable source of
polyphenolic compounds, primarily flavonols, flavanols, phenolic acids, proanthocyanidins, and
anthocyanins [5]. The unique polyphenol composition is responsible for the high biological potential
of elderberry fruit (EDB), including antiviral and antimicrobial activity, as well as chemopreventive,
neuroprotective, and anti-inflammatory effects that have been documented in several scientific
reports [6–10]. Also, it has been suggested that EDB may be an effective remedy for diabetes, obesity,
and metabolic dysfunctions [9]. Animal studies have shown the ability of Sambucus nigra preparations
to improve glucose and lipid metabolism and diabetic osteoporosis status [11–14].
Anthocyanin-rich EDB extract has been proved to attenuate systemic inflammation and insulin
resistance in high-fat diet-induced obese mice. Pro-inflammatory markers of low-grade chronic
inflammation, including serum monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis
factor-α (TNF-α), were significantly reduced in EDB-fed mice. Also, the high-fat diet supplemented
with EDB extract mitigated some metabolic disturbances by lowering serum triglycerides and
improving insulin sensitivity [12]. Lowered insulin resistance was found in diabetic rats fed with
a high-fat diet supplemented with EDB extracts rich in triterpenic acids or polyphenol compounds.
The extracts modulated glucose metabolism by correcting hyperglycemia or reducing insulin
secretion, respectively [13]. The anthocyanin-rich EDB extract protected against inflammation-related
impairments in high-density lipoprotein (HDL) function in a mouse model of hyperlipidemia and HDL
dysfunction. The decrease in total cholesterol content of the aorta in EDB-fed mice suggested limiting
atherosclerosis progression [14]. Scientific reports indicate that EDB extracts possess the unique potential
to modulate the immune response depending on the immune stimuli and inflammatory disorders.
The EDB bioactives have evoked different immune effects by controlling pro- and anti-inflammatory
cytokines and mediators (Reactive oxygen species, NO, IL-6, TNF-α, MCP-1, IL-1, IL-8, IL-10, PGE2 ,
COX-2, iNOS, INF-γ), that play a crucial role in acute and chronic low-grade inflammatory diseases
associated with obesity, diabetes, dyslipidemia, cardiovascular disturbances, and neurodegenerative
diseases [7,8,10–16].
Over the last decade, significant advances in knowledge about the health-beneficial potential of
EDB fruit have been achieved through extensive preclinical studies. However, the results obtained
only in the few clinical trials have not enabled to express an unambiguous opinion and, so far, have
not provided strong evidence of the therapeutic effects of Sambucus nigra fruit in obesity and metabolic
disorders [9]. Recently, the scientific community has stated the need for further research on the
health-promoting properties of this valuable plant as a natural constituent of food products and
beneficial component of a healthy diet [6,9].
This study aimed to evaluate the capacity of Sambucus nigra fruit extract to mitigate obesity-related
metabolic complications through the carbohydrate and lipid metabolism regulation, glucose uptake
improvement, and insulin sensitivity controlling. Also, the goal of the study was the assessment
of the ability of the extract to alleviate the inflammatory response in activated macrophages, which
are recruited into excessively growing fat tissue and may be a primary source of locally produced
pro-inflammatory mediators.
Foods 2019, 8, 326
2.1. Preparation of Elderberry Fruit Extract

The fruits of elderberry (Sambucus nigra L.) cultivar Sampo, obtained from Bio Berry Poland
(Warsaw, Poland), were homogenized to fruit pulp, which was subsequently frozen at −80 ◦ C and
subjected to freeze-drying at a vacuum pressure of 0.1 mbar and temperature of 20 ◦ C for 23 h and
post-drying at 23 ◦ C for 3 h using a freeze dryer (LMC-1, Martin Christ Gefriertrocknungsanlagen
GmbH, Germany). The lyophilized EDB were finely ground and packaged under nitrogen atmosphere.
The EDB extract was obtained by dissolving the EDB powder in complete culture medium with the pH
adjustment to 7.4. The EDB suspension was then centrifuged (3000 g, 5 min) and filtered through a
0.22 μm membrane (Merck, Germany).
2.2. Determination of Individual Phenolic Compounds Using HPLC-DAD-MSn Analysis

Analyses of phenolic compounds were performed on an Agilent 1200 series HPLC system (Agilent
Technologies, Inc., Santa Clara, CA, USA) that was equipped with a G1315D photodiode array detector
and coupled online with an Agilent 6224 time-of-flight MS system. Phenolic compounds were identified
using a mass spectrometer fitted with an electrospray ionization (ESI) source that was operated in
positive-ion or negative-ion mode. Analyses were carried out using full MS scan mode, and full mass
spectra were recorded in the range of 100 to 1700 m/z. Technical specification of apparatus and major
HPLC/MS parameters and analysis conditions were described in detail in our previous work [17].
For quantification purposes, all anthocyanins conjugates were expressed as cyanidin-3-glucoside
equivalents; all flavan-3-ols and their polymers as catechin equivalents; hydroxybenzoic acid glucoside
and hydrolysable tannins as gallic acid equivalents; phenolic acids derivatives as chlorogenic acid
equivalents; and flavonol glycosides as quercetin equivalents.
2.3. T3-L1 Cell Culture, Differentiation, and Treatment

The mouse embryo 3T3-L1 cell line was purchased from the American Type Culture Collection
(ATCC, CL-173). The 3T3-L1 preadipocytes were grown, passaged, and differentiated into adipocytes
as described previously [18]. The 3T3-L1 cells were grown in Dulbecco’s Modified Eagle’s Medium
(DMEM) with 10% calf serum supplementation (Sigma-Aldrich, Merck Group, Darmstadt, Germany).
Cell differentiation was induced in post-confluent cell cultures by a differentiation mixture consisting
of 1 μM insulin, 0.25 μM dexamethasone (DEX), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) in
DMEM with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific Polska, Warsaw, Poland).
Fully differentiated 3T3-L1 cells were exposed to the EDB extract at concentrations of 5, 10, and
20 mg/mL for 24 h. The levels of intracellular ROS generation and lipid accumulation in mature
adipocytes were determined. Also, the viability and metabolic activity of the mature adipocytes were
analyzed after the treatment.
After completion of the differentiation process, insulin resistance was induced in 3T3-L1 adipocytes
by 10 ng/mL murine TNF-α (Sigma-Aldrich) for 5 days, with medium/TNF-α replacement every 2 days.
Glucose uptake measurement was performed in insulin-resistant and insulin-sensitive adipocytes
subjected to the EDB treatment.
2.4. Macrophage Cell Culture and Anti-Inflammatory Experiment Procedure

RAW 264.7 murine macrophage line was obtained from the European Collection of Authenticated
Cell Cultures (ECACC, 91062702) and supplied by Sigma-Aldrich. Cells were grown in DMEM
supplemented with 10% heat-inactivated FBS at 37 ◦ C in a humidified, 5% CO2 , 95% air atmosphere.
The 24-h cultures of RAW 264.7 macrophages, seeded at a density of 5 × 105 cells/cm2 , were treated
with EDB extract prepared in DMEM at the concentrations of 0.1, 1, and 10 μg/mL and incubated for 2 h
in standard culture conditions. Controls were treated with DMEM only. Subsequently, macrophages
were stimulated with 5 ng/mL of lipopolysaccharide (LPS) from Escherichia coli O-127 (Sigma-Aldrich).
Foods 2019, 8, 326
After 3-h macrophage activation, the culture media and cells were harvested to analyze the protein
secretion and gene expression of pro-inflammatory mediators.
2.5. Cell Viability Assay

The viability and metabolic activity of differentiated 3T3-L1 adipocytes and LPS-stimulated RAW
264.7 macrophages were analyzed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) test (Sigma–Aldrich) following the protocol described previously [8].
2.6. Measurement of Reactive Oxygen Species in Adipocytes

The intracellular ROS generation was determined using nitro blue tetrazolium (NBT) according to
the procedure described by Choi et al. [19]. The cells were incubated in 0.2% NBT solution for 90 min,
washed with phosphate-buffered saline (PBS), fixed with methanol, and then air-dried. The formazan
extraction was performed using KOH and DMSO for dissolving. The absorbance was measured at
620 nm using a Tecan M200 Infinite microplate reader (Tecan Group Ltd., Männedorf, Switzerland).
2.7. Measurement of Intracellular Triglyceride Content in Adipocytes

Total concentrations of triglycerides (TG) in differentiated 3T3-L1 adipocytes were determined
using Adipogenesis Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol. Intracellular
TG content was measured by a coupled enzyme assay, which resulted in a fluorometric product
detected at λex = 535 nm and λem = 587 nm (Tecan M200 Infinite), which was proportional to the TG
present. The TG concentration was calculated based on the curve plotted for TG standards.
2.8. Glucose Uptake Measurement in Adipocytes

Glucose uptake assay was performed according to the modified method of Alonso-Castro and
Salazar-Olivo [20]. Mature 3T3-L1 adipocytes, cultured on 24-well plates for fluorescence-based assays,
were starved in serum-free medium (MEM containing BSA 0.5%) overnight. Subsequently, the medium
was replaced with Krebs Ringer phosphate HEPES (KRPH) buffer containing 0.2% BSA (KRPH/BSA)
and incubated for 60 min. The cells were then exposed for 60 min to EDB extract suspended
in KRPH/BSA buffer supplemented with 80 μM 2-NBDG (2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl)
amino-2-deoxy-d-glucose) (Sigma-Aldrich) used as fluorescent glucose analogue. The control cultures
were treated with 100 nM insulin or 10 μM rosiglitazone (Sigma-Aldrich). After incubation, cultures
were immediately washed three times with ice-cold PBS. The fluorescence intensity of 2-NBDG was
measured at λex = 485 nm and λem = 535 nm (Tecan M200 Infinite).
2.9. Determination of Adipokine Production in 3T3-L1 Adipocytes

The leptin and adiponectin concentrations were measured using ELISA kits (Sigma-Aldrich, Merck
Group) following the manufacturer’s instructions. The adipokine concentrations were expressed in
ng/mL of culture medium, which was equivalent to the amount of protein per 1 × 106 cells.
2.10. Determination of IL-6, TNF-α, and PGE2 Production in RAW 264.7 Macrophages
The secretion of IL-6 and TNF-α cytokines as well as generation of PGE2 by LPS-stimulated RAW
264.7 macrophages were determined with ELISA kits (R&D Systems, Inc, Minneapolis, MN, USA)
according to the manufacturer’s instructions. Protein concentrations were expressed in pg/mL of
culture supernatant, which was equivalent to the amount of protein per 1 × 106 cells.
2.11. Determination of NO Production in RAW 264.7 Macrophages

Griess method was applied to determine nitrite as an indicator of NO production. Equal volumes
of the Griess reagent (Sigma-Aldrich) and RAW 264.7 culture supernatant were mixed and incubated
Foods 2019, 8, 326
at room temperature for 15 min. The absorbance was measured at 540 nm (Tecan M200 Infinite). The
standard curve plotted for sodium nitrite was used to calculate NO concentration.
2.12. Quantification of Gene Expression Using Real-Time PCR

The analysis of gene expression was carried out in accordance with the detailed protocol presented
in the previous work [17]. The TRI reagent (Sigma-Aldrich) was used to isolate total RNA, Synthesis
cDNA Transcriptor First-Strand kit (Roche Diagnostics GmbH, Mannheim, Germany) for first-strand
cDNA synthesis, and SYBR1 Select Master Mix (Life Technologies, Carlsbad, CA, USA) for real-time
PCR. The primers used for the amplification of cDNAs are listed in Table 1.
Table 1. The primers sequence used for real-time PCR.
Gene Accession No. Sequence (5 –3 ) Amplicon (bp)

F: GGA TCA GGT TTT GTG GTG CT
Mm LEP NM-008493 187
R: TTG TGG CCC ATA AAG TCC TC
F: TGC TGG GCA CAG CTA CCC
Mm GLUT-4 NM-001359114.1 162
R: CGG TCA GGC GCT TTA GAC
F: CTG GCC ACT TTC TCC TCA TT TC
Mm ADIPOQ NM-009605 120
R: GGC ATG ACT GGG CAG GAT TA
F: TCT GAA GGA CTC TGG CTT TG
Mm IL-6 NM-031168.1 142
R: GAT GGA TGC TAC CAA ACT GGA
F: TGA AGA AAA CCC CTT GTG CT
Mm NOS-2 NM-010927.3 100
R: TTC TGT GCT GTC CCA GTG AG
F: GGC GCA GTT TAT GTT GTC TGT
Mm PTGS2 NM-011198.3 107
R: CAA GAC AGA TCA TAA GCG AGG A
F: AGG GTC TGG GCC ATA GAA CT
Mm TNF-α NM-001278601.1 103
R: CCA CCA CGC TCT TCT GTC TAC
F: GAT CAC AGA AGG TCC CTA GCA G
Mm NOX-4 NM-015760.5 134
R: GTT GAG GGC ATT CAC CAA GT
F: CGT GTC TGT GGG AGT CCA AGG TTC AG
Mm SOD2 NM-013671.3 139
R: GTC AAT CCC CAG CAG CGG AAT AAG
F: CCT CCT CGT TCA GGA TGT GGT T
Mm CATALASE NM-009804.2 243
R: CGA GGG TCA CGA ACT GTG TCA G
F: GGG CAA GGT GCT GCT CAT TG
Mm GPx NM-008160.6 269
R: AGA GCG GGT GAG CCT TCT CA
F: CCA CAG CTG AGA GGG AAA TC
Mm ACTB NM-007393 193
R: AAG GAA GGC TGG AAA AGA GC
The relative expression of each gene was calculated using the 2−ΔΔCT method. The mRNA levels
in the control cells were designated as 1, and the relative levels of the gene transcripts in the samples
were expressed as the fold change.
2.13. Digestive Enzyme Inhibition Assays
2.13.1. Measurement of Pancreatic Lipase Inhibition

The EDB inhibitory activity against pancreatic lipase (EC 3.1.1.3) was evaluated according to
the method of Boath et al. with minor modification [21]. The p-nitrophenyl laurate (pNP laurate)
was used as a substrate. The pNP laurate was dissolved to 0.08% in 5 mM sodium acetate (pH 5.2)
containing 1% Triton X-100 and 0.05% Arabic gum. The reaction mixture consisting of 350 μL of assay
buffer (100 mM Tris, pH 8.2), 50 μL of EDB extract, 150 μL of pancreatic lipase type II from porcine
pancreas (10 mg/mL), and 450 μL of substrate solution was incubated at 37 ◦ C for 2 h. Orlistat, a known
porcine pancreatic lipase inhibitor, was applied as a positive control. After incubation, the sample was
centrifuged at 13,000 rpm for 3 min and read at 400 nm of wavelength (Tecan M200).
Foods 2019, 8, 326
2.13.2. Measurement of α-Amylase Inhibition

The inhibition of α-amylase (EC 3.2.1.1) activity was determined using the method of Tan et al.
with slight modification [22]. The reaction mixture consisting of 200 μL of distilled water, 50 μL of
EDB extract, 250 μL of α-amylase from porcine pancreas (30 mg/mL), and 500 μL of 0.5% starch was
incubated at 37 ◦ C for 10 min. Acarbose, a known pancreatic α-amylase inhibitor, was applied as a
positive control. Enzymatically released reducing sugars were determined by DNS reagent solution
(96 mM 3,5-dinitrosalicylic acid, 5.31 M sodium potassium tartrate in 2 M NaOH) after heating at 95 ◦ C
for 10 min. Then, the mixture was diluted with distilled water and the absorbance was measured at
540 nm (Tecan M200 Infinite).
2.13.3. Measurement of α-Glucosidase Inhibition

The inhibition assay of α-glucosidase (EC 3.2.1.20) was adopted from Tan et al. [22]. The
p-nitrophenyl-α-d-glucuronide (pNPG) dissolved to 4 mM in 0.1 M HEPES (pH 6.8) was used as a
substrate. The reaction mixture consisting of 350 μL of HEPES (pH 6.8), 50 μL of EDB extract, 150 μL
of α-glucosidase (20 mg/mL), and 450 μL of substrate solution was incubated at 37 ◦ C for 2 h. The
release of p-nitrophenol from the pNPG substrate was measured at 410 nm (Tecan M200 Infinite). As a
positive control, the glucosidase inhibitor, acarbose, was used.
All reagents used in digestive enzyme inhibition assays were provided by Sigma-Aldrich.
2.13.4. Data Analysis

Enzyme activity in the presence of inhibitor (EDB extract or reference inhibitor) was expressed as
a percentage of the non-inhibited enzyme activity and plotted versus inhibitor concentration. Based on
the dose-response curve, the inhibitor concentration required for 10% and 50% inhibition of enzyme
activity (IC10 and IC50 ) was determined as a measure of inhibitory potency. The percentage of the
non-inhibited enzyme activity was calculated by following equation:
% non-inhibited enzyme activity = [(AInhibitor − AInhibitor blank )/(AControl − AControl blank )] × 100%
where AControl is the absorbance of the sample without EDB extract/reference inhibitor; AInhibitor is the
absorbance of the sample containing EDB extract/reference inhibitor; AInhibitor blank is the absorbance
of the sample with EDB extract/reference inhibitor, but without enzyme addition; AControl blank is the
absorbance of the sample without EDB extract/reference inhibitor and enzyme addition.

All data are expressed as the means ± SD from three independent experiments. Statistical analysis
was performed using the STATISTICA version 13.3 software (Statsoft, Inc., Tulsa, OK, USA). One-way
analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to determine the differences
between the mean values of multiple groups. The T-student’s test was applied to determine the
significant difference between two independent groups. The equality of variances assumption was
verified with the Levene’s test.
3. Results
3.1. Polyphenol Composition in the Elderberry Fruit Extract

HPLC-DAD-ESI-MSn analysis of the EDB extract revealed the presence of 22 polyphenolic
compounds, including anthocyanins (peaks 1–5), hydroxybenzoic acid derivative (peak 6), flavan-3-ols
(peaks 7–8), polymers, tentatively identified as hydrolysable tannins (peaks 9–10), hydroxycinnamic
acids (peaks 11–15), and flavonols (peaks 16–22). HPLC-DAD chromatograms and chromatographic
characteristics with mass spectral data of polyphenols identified in the EDB extract are presented in
Figure 1 and Table 2, respectively.
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Table 2. HPLC-MS identification of phenolic compounds in elderberry fruit extract in positive

(electrospray ionization (ESI) +) and negative (ESI −) ionization mode.
UV λ max [M]+ /[M + [M − H]− MS/MS Concentration

Peak No. RT (min) Tentative Identification
(nm) H]+ (m/z) (m/z) (m/z) (mg/g) *
611.1651 - 287.0583 Cyanidin-3,5-O-diglucoside
1 14.01 280, 520 3.27 ± 0.25
Cyanidin-3-O-sambubiosyl-5-O-
743.2095 287.0579
glucoside (co-elution)
2 16.90 280,520 449.1133 287.0632 Cyanidin-3-O-glucoside Trace amounts
3 19.84 280,520 595.1734 287.0578 Cyanidin-3-O-rutinoside Trace amounts
4 23.92 280,520 433.1187 271.0640 Pelargonidin-3-O-glucoside 0.31 ± 0.04
5 29.85 280,520 581.1635 287.0633 Cyanidin-3-O-sambubioside 9.76 ± 0.68
6 4.48 275 299.2506 — 4-Hydroxybenzoic acid glucoside 1.60 ± 0.12
7 9.05 280 289.1139 245.1203 (+)/(−)-Catechin 1.07 ± 0.06
8 9.99 280 289.1140 245.1210 (+)/(−)-Epicatechin 1.41 ± 0.08
9 13.96 268 597.4616 — Hydrolysable tannin 3.45 ± 0.22
10 14.38 268 597.4628 — Hydrolysable tannin 0.92 ± 0.06
11 9.99 299,325 353.2873 191.1737 Neochlorogenic acid 0.50 ± 0.03
12 11.00 299,325 353.2886 191.1749 Chlorogenic acid 0.59 ± 0.04
13 12.54 300,325 353.2865 191.1729 Cryptochlorogenic acid 0.26 ± 0.02
14 17.00 310,234 337.0917 173.0443 P-coumaroylquinic acid 0.78 ± 0.06
15 17.52 316,234 371.3016 163.0396 P-Coumaric acid hexoside 2.85 ± 0.18
16 22.83 268,354 463.0882 301.0354 Quercetin-3-O-glucoside 0.29 ± 0.01
17 24.39 255,355 609.1461 301.0349 Quercetin-3-O-rutinoside 2.27 ± 0.19
Quercetin
18 28.63 255,358 505.0872 301.0366 0.09 ± 0.01
3-O-(6”-acetyl-glucoside)
19 29.02 266,348 447.0935 285.0540 Kaempferol-3-O-glucoside 0.09 ± 0.01
20 30.34 255,352 593.1515 285.0542 Kaempferol-3-O-rutinoside 1.36 ± 0.12
21 33.15 255,370 301.0356 151.0031 Quercetin 0.09 ± 0.02
22 36.14 255,352 623.1044 315.0449 Isorhamnetin-3-O-rutinoside 0.07 ± 0.01
* mg/g of lyophilized elderberry powder, values were expressed as mean ± SEM for three independent experiments.
Anthocyanins accounted for 43% of all polyphenolics; cyanidin-based anthocyanin

compounds (-3,5-O-diglucoside, -3-O-sambubiosyl-5-O-glucoside, -3-O-glucoside, -3-O-rutinoside,
and -3-O-sambubioside) were the main group of anthocyanins, with a significant predominance
of cyanidin-3-O-sambubioside ([M + H]+ at m/z 287) constituting 73.2% of all anthocyanins. Peak
1 contained two compounds, identified as cyanidin-3,5-O-diglucoside ([M + H]+ at m/z 611) and
cyanidin-3-O-sambubiosyl-5-O-glucoside ([M + H]+ at m/z 743). These anthocyanins represented
25.0% of total anthocyanin compounds, quantitatively determined in the EDB extract. In contrast,
cyanidin-3-O-glucoside ([M + H]+ at m/z 449) and cyanidin-3-O-rutinoside ([M + H]+ at m/z 595) were
only detected in trace amounts in the EDB extract (Table 2). The anthocyanin with a molecular ion of
m/z 433, that yielded on MS2 fragment at m/z 271, was identified as pelargonidin-3-O-glucoside and
quantified in small amounts estimated at 2.3% of all anthocyanins.
Other groups of compounds: Flavan-3-ols, hydroxycinnamic acids, and flavonols amounted to
22.1%, 16.0%, and 13.7% of the total content of polyphenols, respectively. Moreover, the presence
of 4-hydroxybenzoic acid glucoside (5.2%) was found in the extract. The group of non-anthocyanin
compounds with the largest share in the polyphenol pool were flavan-3-ols, among which catechin
and epicatechin (36%), and tannins (64%) were identified. A total of five compounds were detected
within another group of hydroxycinnamic acid derivatives, including p-coumaric acid hexoside (57%),
p-coumaroylquinic acid (16%), chlorogenic acid, and its isomers: Neochlorogenic and cryptochlorogenic
acids (27%). Concerning flavonols, the results of the HPLC-DAD analysis revealed the presence
of quercetin, kaempferol, and isorhamnetin derivatives, with quercetin-3-O-rutinoside (53%) and
kaempferol-3-O-rutinoside (32%) quantified as the dominant compounds within this class.
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Figure 1. Cont.
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Figure 1. HPLC-DAD chromatograms of elderberry (Sambucus nigra L.) fruit extract recorded at 520, 280, 325, and 355 nm, respectively. Peak numbers and retention
times refer to compounds indicated in Table 2.
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The polyphenolic compounds, described above, have been previously identified in berries
of Sambucus nigra [7,23,24]. However, the content of individual polyphenols in EDB fruits varies,
depending on the EDB genotypes as well as specific growth conditions. The total polyphenol content
in the extract analyzed was determined to 31.03 mg/g of EDB lyophilized powder, including 13.34 mg
of anthocyanins, 6.85 mg of flavan-3-ols, 4.98 mg of hydroxycinnamic acid derivatives, 4.26 mg of
flavonols, and 1.6 mg of hydroxybenzoic acid glucoside (Table 2).
3.2. Digestive Enzyme Activity Inhibition by Elderberry Fruit Extract

The EDB extract was evaluated for the ability to inhibit digestive enzymes, including α-glucosidase,
α-amylase and lipase. The extract showed similar α-glucosidase and α-amylase inhibitions (Figure 2a,b),
with the same IC10 value of 1.25 mg/mL and non-significantly different IC50 values of 6.38 mg/mL and
6.70 mg/mL, respectively (Table 3).
Figure 2. The effect of elderberry fruit extract (EDBE) on the activity of digestive enzymes: α-glucosidase
(a), α-amylase (b), and lipase (c). Enzyme activity is expressed in relation to the negative control
(without extract addition). As positive controls, reference enzyme inhibitors, including acarbose (ACRB,
100 μg/mL) and orlistat (ORST, 5 μg/mL) were used in the experiment. The values represent the means
(n = 3) ± SD. * p < 0.05, *** p < 0.001 vs. control group.
Table 3. Inhibitory concentrations IC10 and IC50 (the inhibitor concentration required for 10% and 50%
inhibition of enzyme activity) of the elderberry fruit extract (EDBE), acarbose (ACRB), and orlistat
(ORST) as reference pharmacological inhibitors.
Enzyme α-Glucosidase α-Amylase Lipase

Inhibitor IC10 IC50 IC10 IC50 IC10 IC50
EDBE (mg/mL) 1.25 ± 0.09 6.70 ± 0.56 1.25 ± 0.10 6.38 ± 0.44 2.09 ± 0.30 10.98 ± 0.47
ACRB (μg/mL) 85.12 ± 0.63 > 100 1.30 ± 0.06 8.23 ± 0.16 - -
ORST (μg/mL) - - - - 0.2 ± 0.02 1.43 ± 0.15
Lower potency of the extract was observed in inhibiting pancreatic lipase activity. The extract
concentrations reducing lipase activity by 10% and 50% were determined at 2.09 mg/mL and
10.98 mg/mL, respectively (Table 3). In inhibiting α-amylase and lipase activity, the drugs acarbose
(100 μg/mL) and orlistat (5 μg/mL) were more potent than the EDB extract (Figure 2b,c). However, in
α-glucosidase inhibition, the extract at concentrations of 5 mg/mL and 10 mg/mL evoked the stronger
effects than acarbose at a dose of 100 μg/mL.
3.3. The Effect of Elderberry Fruit Extract on Hypertrophied Adipocytes

To determine whether EDB extract affects the condition of mature adipocytes, we examined
its effect on the viability, lipid accumulation, and ROS production, as well as regulation of leptin
and adiponectin expression in terminally differentiated 3T3-L1 cells. The obtained results indicated
that EDB extract did not exert any significant effect on both adipocyte viability (Figure 3a) and
intracellular lipid content (Figure 3b,g–i). Nevertheless, it dose-dependently inhibited the intracellular
ROS generation in hypertrophied adipocytes. EDB extract caused 36%, 53%, and 58% decrease in ROS
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level when applied at 5, 10, and 20 mg/mL, respectively, in comparison to untreated cells (p < 0.001)
(Figure 3c). Moreover, treatment with EDB extract led to down-regulation of NADPH oxidase 4 (NOX-4)
mRNA expression by approximately 49 ± 6%, independently of the extract dose. Treatment with the
extract at the maximum concentration (20 mg/mL) resulted in approximately 2-fold increased mRNA
expression of superoxide dismutase (SOD) and glutathione peroxidase (GPx). In contrast, the extract did
not influence the mRNA expression level of catalase (CAT) (Figure 3d).
Figure 3. Changes in cell viability (a), intracellular triglyceride content (b), reactive oxygen species
production (c), and mRNA expression of NOX4, SOD2, catalase (CAT), and GPx enzymes (d) as well
as leptin (LEP) (e) and adiponectin (ADIPOQ) (f) expression upon the treatment of3T3-L1 mature
adipocytes with the elderberry fruit extract (EDBE). The photos present 3T3-L1 preadipocytes (g),
fully differentiated 3T3-L1 adipocytes non-treated (h) and treated with EDBE at the concentration of
20 mg/mL (i). The cells were photographed at magnification of 100 ×. The results were expressed as
the means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group.
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Supplementation of the fully differentiated adipocyte cultures with EDB extract considerably
affected the expression of key adipokines in treated adipocytes. The decrease in leptin (LEP) mRNA
levels between 78% and 94% was observed in mature adipocytes exposed to the extract at concentrations
ranging from 5 mg/mL to 20 mg/mL (p < 0.001) (Figure 3e). In contrast to leptin, the expression of
adiponectin was significantly up-regulated. The highest dose of EDB extract elevated adiponectin
(ADIPOQ) mRNA level by 77% compared to the control (p < 0.001) (Figure 3f). The exposure of mature
adipocytes to EDB extract resulted in a decrease of leptin secretion. The extract at concentrations
of 5 mg/mL and 10 mg/mL significantly reduced leptin synthesis (p < 0.05). Whereas, the highest
inhibitory effect with the reduction of leptin by 86% (p < 0.001) was observed in the cells treated
with the extract at maximum concentration. In contrast, EDB extract at the highest dose of 20 mg/mL
stimulated the adiponectin secretion in treated cells. The level of adiponectin was increased by 36%
respect to the control (p < 0.05) (Figure 3f).
3.4. The Effect of Elderberry Fruit Extract on Glucose Uptake in Mature 3T3-L1 Adipocytes
The effect of EDB extract on the glucose analogue (2-NBDG) uptake in mature 3T3-L1 adipocytes
was analyzed to determine whether the extract affects the glucose uptake by adipocytes. As shown in
Figure 4a, EDB extract caused a significant increase in 2-NBDG uptake at all assayed concentrations
(p < 0.001). The extract stimulated 2-NBDG uptake by 40%, 44%, and 62% tested at 5, 10, and 20 mg/mL,
respectively, in comparison to the control system. Unexpectedly, the stimulatory effect of the extract on
the glucose uptake in insulin-sensitive cells was more effective than that observed with rosiglitazone
(Figure 4a). Subsequently, the expression of glucose transporter type 4 (GLUT-4) gene in mature 3T3-L1
cells exposed to EDB extract was investigated. The results indicated no significant effect of EDB extract
on GLUT-4 mRNA level (Figure 4d).
Figure 4. Effect of the elderberry fruit extract (EDBE) on the glucose uptake (a–c) and glucose transporter
GLUT-4 mRNA expression (d–f) in mature 3T3-L1 adipocytes non-induced (a,d) and induced by TNF-α
(b–f) and non-treated (b,e) and treated (c,f) with insulin (INS). The cells were exposed to EDBE at
concentrations of 5, 10, and 20 mg/mL, and to rosiglitazone (RGZ). Data are mean values ± SD (n = 3).
The significance of the main effects of EDBE was determined by Tukey post hoc test; the control (INS,
RGZ) significance was analyzed by T-student test; * p < 0.05, ** p < 0.01, *** p < 0.001.
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In the parallel experiment, the influence of EDB extract on 2-NBDG incorporation into insulin
resistant adipocytes treated with TNF-α was determined. The cells were incubated with the extract
in the absence or presence of insulin at 100 nM. In the insulin-resistant adipocytes cultured without
insulin supplementation, the EDB extract at concentrations of 5, 10, and 20 mg/mL enhanced 2-NBDG
uptake by 40%, 38%, and 39%, respectively (Figure 4b). In this case, quantitative PCR analysis revealed
that treatment with EDB extract at doses from 5 mg/mL to 20 mg/mL significantly up-regulated the
mRNA expression of GLUT-4 with an increase ranging from 63% to 82% (Figure 4e).
In the experiments on insulin resistant adipocytes exposed to insulin, the EDB extract at the
highest concentration showed insulin-sensitizing properties, stimulating 2-NBDG incorporation by
34% (p < 0.01). Its efficacy was comparable to that of rosiglitazone, which enhanced the 2-NBDG
uptake by 36% (p < 0.01) (Figure 4c). In this study, the extract at a dose of 20 mg/mL up-regulated the
expression of GLUT-4 by 33% (p < 0.01), compared to control adipocytes (Figure 4f).
3.5. Anti-Inflammatory Effects of Elderberry Fruit Extract

Anti-inflammatory effects of EDB extract were evaluated in activated RAW 264.7 macrophages,
considering proinflammatory cytokines and mediators determined at both molecular and cellular
levels, using low non-cytotoxic extract doses of 0.1, 1, and 10 μg/mL. The obtained results demonstrated
the ability of EDB extract to alleviate the cellular inflammatory response induced by LPS. The extract
at concentrations of 1 and 10 μg/mL suppressed mRNA expression of IL-6 by 34% (p < 0.01) and 69% (p
< 0.001), respectively, compared to control macrophages. Moreover, a 28% decrease in TNF-α mRNA
level (p < 0.05) was observed following treatment with EDB extract at a dose of 10 μg/mL (Figure 5a).
The down-regulation of IL-6 and TNF-α expression was consistent with the inhibited secretion of these
cytokines. Namely, EDB extract assayed at 1 μg/mL reduced production of IL-6 and TNF-α by 44%
and 26%, respectively (p < 0.05). At the extract concentration of 10 μg/mL, the 60% decrease in IL-6
secretion (p < 0.01) was observed while synthesis of TNF-α was reduced by 52% (p < 0.001) (Figure 5b).
Figure 5. Effect of the elderberry fruit extract (EDBE) on mRNA expression of IL-6, TNF-α, COX-2, and
iNOS (a) and on the production of IL-6, TNF-α, PGE2 protein, and NO (b) in the lipopolysaccharide
(LPS)-activated RAW 264.7 macrophages. Data are mean values ± SD (n = 3). * p < 0.05, ** p < 0.01,
*** p < 0.001 when compared with control.
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The extract at a dose of 10 μg/mL also affected COX-2 expression, causing a 46% reduction of
COX-2 transcripts level in activated macrophages (p < 0.01) (Figure 5a). As a consequence, the decrease
in PGE2 production was detected after cell exposure to the extract. Namely, PGE2 level lowered by
33% and 47%, respectively, for a dose of 1 μg/mL and 10 μg/mL (Figure 5b).
Furthermore, the obtained results demonstrated the reduction in NO production as well as
down-regulation of inducible NO synthase (iNOS) expression in LPS-stimulated RAW 264.7 macrophages
in response to the EDB extract treatment. The extract reduced the NO synthesis by 35% (p < 0.05)
when assayed at 1 μg/mL and by 54% (p < 0.01) when applied at 10 μg/mL (Figure 5b). A significant
inhibitory effect of EDB extract on iNOS expression was noted only at the highest tested dose, which
decreased iNOS mRNA level by 30% (p < 0.01) (Figure 5a).
4. Discussion
Excessive fat accumulation in hypertrophic adipose tissue associated with obesity is responsible
for oxidative stress, chronic inflammation, and dysregulated adipokine secretion [25]. It is believed that
the therapeutic potential of natural dietary compounds against obesity and obesity-related disorders
should focus on improving the fat function in pathogenic hypertrophic adipocytes by reducing
oxidative stress, alleviating inflammation, and regulating underproduction or overproduction of
clinically relevant adipocyte factors. However, most bioactive compounds or extracts strongly affect
preadipocytes, their viability, proliferation, and differentiation into mature fat cells, without any
significant effects on the pathological status of hypertrophic adipocytes. Therefore, in this work, the
influence of the Sambucus nigra fruit extract on mature fully differentiated insulin-resistant 3T3-L1
adipocytes was investigated.
In our study, we found no reduction in cell viability and lipid content in hypertrophic 3T3-L1
adipocytes after exposure to EDB extract. However, as a result of the treatment, the intracellular ROS
generation was significantly down-regulated and probably, oxidative stress accompanying excessive
fat accumulation was also importantly reduced. Oxidative stress induced by enhanced lipid content is
reported to be involved in the pathogenesis of obesity-related comorbidities including insulin resistance
and diabetes, cardiovascular complications, and cancer [26]. It was found that ROS are intensively
generated in visceral adipose tissue by adipocytes during the metabolism of excess nutrients and also
by macrophages, which accumulate in adipose tissue in obesity state. The increased release of fatty
acids from overproduced fat accumulated in adipose tissue, activate NADPH oxidases (NOX) and
induce or aggravate ROS production. Other factors that also contribute oxidative stress to obesity
include hyperleptinemia, low antioxidant defense, or chronic inflammation [27]. Our results showed
that EDB extract could reduce ROS generation by lowering the expression of NOX4, the major NOX
isoform in adipocytes. Treatment of hypertrophied 3T3-L1 adipocytes with EDB extract caused a
significant decreasing in NOX4 mRNA expression. Furthermore, up-regulation of mRNA expression
of antioxidant enzymes, like SOD and GPx, could also contribute to enhancing adipocyte antioxidant
defense efficiency. Numerous studies have shown the high antioxidant capacity of Sambucus nigra
fruit [6,10]. However, the antioxidant effects of EDB on adipocytes have not yet been reported in the
literature. In the present study, we demonstrated that introduction of EDB extract to the culture of
hypertrophic adipocytes resulted in decreased ROS generation in cells. The antioxidant action of EDB
extract in adipocytes may be a potential protective mechanism against obesity-associated pathological
risk factors, including insulin resistance and chronic inflammation.
Additionally, EDB extract treatment modulated the leptin and adiponectin gene expression and
protein secretion in hypertrophic 3T3-L1 adipocytes. Leptin and adiponectin are adipocytokines, which
influence energy homeostasis, glucose and lipid metabolism, cardiovascular function, and immune
response [28]. Leptin is primarily secreted by fully differentiated adipocytes, and its crucial role is to
regulate energy intake and expenditure through controlling appetite and glucose metabolism. Reflecting
the increased amount of adipose tissue, obese individuals often have elevated leptin concentration
and the simultaneous apparent loss of efficacy of leptin, which is a result of leptin resistance, the
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state that leads to uncontrolled food intake, pro-inflammatory state, diabetes mellitus, and other
obesity-related complications [29]. In contrast to leptin, adiponectin is down-regulated in obesity, and
the circulating adiponectin levels are inversely correlated with body fat amount. Adiponectin enhances
energy metabolism and fatty acid oxidation, promotes insulin sensitivity, improves glucose tolerance,
and exerts anti-inflammatory effects [28]. Low serum adiponectin and high serum leptin levels are
considered as risk factors for developing type 2 diabetes (T2DM), obesity, dyslipidemia, hypertension,
and cardiovascular diseases. In this study, a remarkable decrease in leptin expression and secretion was
observed in response to EDB extract treatment of hypertrophied 3T3-L1 adipocytes, which may help
counteract the leptin resistance state. Whereas, adiponectin mRNA expression and protein secretion in
treated adipocytes were significantly increased. The effect of EDB extract on adiponectin production
may indicate anti-inflammatory potential and insulin-sensitizing activity of Sambucus nigra fruit.
The association of visceral obesity with T2DM is a long-recognized phenomenon. The primary
determinant of this correlation is the fact that central obesity is the critical factor in the emergence of
insulin resistance. The insulin-resistant state results in defective insulin-stimulated glucose uptake and
consequently in hyperglycemia, elevated circulating free fatty acids level, abnormal fat accumulation,
and dysregulation of hepatic glucose production, that, in combination with a paucity of insulin secretion
by pancreatic β-cells, leads to T2DM [30]. These metabolic abnormalities may arise from impairment
in insulin signaling pathways and subsequent defect in translocation of insulin-responsive glucose
transporter protein (GLUT-4) and in adipose tissue, also from down-regulation of GLUT-4 gene [31].
The effects of EDB extract on glucose uptake and GLUT-4 expression were evaluated in this
study. Experiments were performed both with mature 3T3-L1 adipocytes sensitive to insulin and
adipocytes treated with TNF-α to induce an inflammatory status and insulin resistance. Analysis
revealed that EDB extract stimulated the 2-NBDG uptake in both types of adipocytes and up-regulated
mRNA expression of GLUT-4 in insulin-resistant cells, suggesting insulin-like and insulin-sensitizing
activities of the extract. The signaling pathways involved in the development of these activities will
be further examined in future studies. This is the first study assessing the effects of EDB extract on
glucose uptake in 3T3-L1 cells. Although several recent reports have suggested the anti-diabetic and
hypoglycemic properties of elderberry, it has been found that EDB methanolic extracts markedly
stimulate glucose uptake in liver HepG2 cells and also exert inhibitory effect towards carbohydrate
hydrolyzing enzyme [32]. Furthermore, EDB extracts, EDB anthocyanins, mainly cyanidin-3-glucoside
and cyanidin-3-sambubioside, procyanidins, and their metabolites were found to enhance glucose
uptake in human skeletal muscle cells [33]. Whereas, EDB lipophilic and polar extracts were reported
to modulate glucose metabolism or lower insulin secretion contributing to the mitigation of insulin
resistance in T2DM rats [13].
Anti-obesity and anti-diabetic activity of EDB extract could be related to the inhibition of dietary
fat and sugar absorption from the intestinal tract. There is some evidence that polyphenols from
berry fruits, such as strawberry, raspberry, blueberry, bilberry, black and red currant, lingonberry, red
and green gooseberry, cranberry, and chokeberry, contribute to the inhibition of digestive enzymes
involved in the hydrolysis of dietary lipids and carbohydrates [34]. Based on our research, the Sambucus
nigra fruit may be included in the class of berries considered as effective inhibitors of α-amylase,
α-glucosidase, and pancreatic lipase activity.
Obesity is known to be accompanied by metaflammation—low-grade chronic inflammation
condition triggered by excess nutrients in metabolic cells [35]. An attribute of obesity-related
inflammation is enhanced infiltration of macrophages into expanding adipose tissue, activation
of specialized immune cells, and secretion of proinflammatory cytokines such as TNF-α, IL-6,
and MCP-1 leading to an unresolved inflammatory response, which affects normal metabolism
and insulin action [35]. Inhibition of obesity-induced inflammation could, thus, be a therapeutic
intervention against adipose tissue dysfunction and related co-morbidities. In recent years, the use of
anti-inflammatory nutrients provided through diet as a potential approach against obesity has been
extensively studied [36,37].
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In the present study, we evaluated anti-inflammatory effects of EDB extract in LPS-stimulated

RAW 264.7 macrophages. Activated macrophages produce cytokines such as TNF-α, IL-1β, and
IL-6 as well as pro-inflammatory mediators, such as NO and PGE2 [38]. IL-6 and TNF-α are potent
proinflammatory cytokines, which play a central role in inflammatory response and are characterized
by a broad spectrum of functions with various effects in adipose tissue. TNF-α substantially influences
lipid metabolism and adipocytes apoptosis. It can disrupt insulin signaling pathway promoting insulin
resistance and adipocytes dysfunction [39]. TNF-α has, thus, been believed to be the crucial mediator
in the detrimental paracrine loop between adipocytes and macrophages [40]. IL-6 has a pivotal role in
acute phase reactions. It also influences hormonal balance and energy homeostasis and may affect the
increase of free fatty acids level. Circulating levels of IL-6 and TNF-α are elevated in obese individuals
and patients with insulin resistance [41]. In general, the regulation of TNF-α and IL-6 secretion is
considered to be a potent treatment strategy for inflammation-associated diseases [42].
The research presented in this work suggests that EDB extract dose-dependently down-regulates
mRNA expression and protein production of TNF-α and IL-6 in activated RAW 264.7 macrophages and
therefore alleviates the cellular inflammatory response induced by LPS. In addition to TNF-α and IL-6,
EDB extract significantly reduced the production of inflammatory mediators—PGE2 and NO. Increased
level of PGE2 is observed in obese adipose tissue due to remarkable up-regulation of COX-2—the
key enzyme in eicosanoid metabolism, of which expression is induced in inflammation state [43].
It has been suggested that COX-2-mediated inflammation in visceral fat is responsible for insulin
resistance and fatty liver development in high-fat-induced obese rats [44]. The same study revealed that
COX-2 inhibition significantly reversed adipocyte hypertrophy, macrophage infiltration, and decreased
markers of adipocyte differentiation. Nitric oxide formed by iNOS is a short-lived vasodilator that acts
as an important regulator of physical homeostasis, while its overproduction has been closely correlated
with the pathological conditions including septic shock, osteoporosis and rheumatoid arthritis, insulin
resistance, and inflammation [45]. In the present study, EDB extract was found to suppress PGE2
and NO production via down-regulation of COX-2 and iNOS expression. These findings indicate
that inhibition of PGE2 and NO generation is one of the anti-inflammatory mechanisms of the extract.
Several recent studies have shown the anti-inflammatory potential of EDB fruit preparations. In our
previous study, we demonstrated the anti-inflammatory potential of gastrointestinally digested EDB
extract following intestinal absorption in a co-culture model of intestinal epithelial Caco-2 cells and
LPS-stimulated RAW 264.7 macrophages [7]. The analyzed extract down-regulated the expression of
genes (IL-1β, IL-6, TNF-α, COX-2) involving in the inflammatory pathway in a range comparable to
that of budesonide. This study demonstrated adequate bioavailability and intestinal permeability of
EDB compounds that are probably sufficient to evoke systemic anti-inflammatory effects [7]. Moreover,
there is increasing evidence that the EDB bioactives can penetrate the blood–brain barrier and modulate
the immune response induced in different types of brain injuries, including ischemic stroke. It has
been found that EDB extract and its phenolic components significantly inhibit activation of microglia,
considered to be resident macrophages responsible for the initial immune response to brain injuries.
Treatment of activated microglial bv-2 cells with EBD extract led to diminishing ROS and NO generation,
and as a consequence, attenuating the neuroinflammatory process [16].
Results of the study, as discussed above, indicate that Sambucus nigra fruit extract may offer
substantial preventive and therapeutic potential for the treatment of obesity and obesity-related
disorders, accompanied by oxidative stress, inflammationm and insulin resistance. Moreover, the
extract can inhibit digestive enzyme activity, and consequently, significantly reduce the intestinal
absorption of dietary lipids and carbohydrates, which is an effective strategy for the prevention and
treatment of obesity and metabolic comorbidities.
Considering the findings of in vitro studies, we can postulate a nutraceutical application of
the Sambucus nigra fruit extract. The scientific community focuses great attention on introducing
nutraceuticals into the daily diet to prevent the occurrence of the pathological conditions, to delay
or avoid the need for drug treatment and to support pharmacological therapy. Nutraceuticals as
Foods 2019, 8, 326
pharmafoods should be evaluated in the clinical aspects regarding safety, side effects, bioavailability,
beneficial health effects, mechanisms of action and efficacy, and any possible interactions between
food and drugs assumed together with them [46,47]. Thus, the developing of clinical studies will
be of significant importance for clinically justified promotion of the Sambucus nigra fruit extract
as a safe nutraceutical with the capacity of prevention or treatment of obesity and obesity-related
immune-metabolic disorders.
Author Contributions: Conceptualization, J.Z.-W. and A.O.; methodology, J.Z.-W., A.O., M.O. and K.K.; formal
analysis, A.O. and R.D.; investigation, J.Z.-W. and M.O.; resources, R.D.; writing—original draft preparation,
J.Z.-W.; writing—review and editing, A.O.; project administration, A.O.; funding acquisition, A.O.
Funding: This research was funded by THE NATIONAL SCIENCE CENTRE, POLAND, grant number
2015/19/B/NZ9/01054.
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foods
Article
Fermented Sea Tangle (Laminaria japonica Aresch)
Suppresses RANKL-Induced Osteoclastogenesis by
Scavenging ROS in RAW 264.7 Cells
Jin-Woo Jeong 1 , Seon Yeong Ji 2,3 , Hyesook Lee 2,3 , Su Hyun Hong 2,3 , Gi-Young Kim 4 ,
Cheol Park 5 , Bae-Jin Lee 6 , Eui Kyun Park 7 , Jin Won Hyun 8 , You-Jin Jeon 4 and
Yung Hyun Choi 2,3, *
1 Nakdonggang National Institute of Biological Resources, Sangju 37242, Korea
2 Department of Biochemistry, Dong-eui University College of Korean Medicine, Busan 47227, Korea
3 Anti-Aging Research Center, Dong-eui University, Busan 47227, Korea
4 Department of Marine Life Sciences, Jeju National University, Jeju 63243, Korea
5 Department of Molecular Biology, College of Natural Sciences, Dong-eui University, Busan 47340, Korea
6 Marine Bioprocess Co. Ltd., Busan 46048, Korea
7 Department of Oral Pathology and Regenerative Medicine, School of Dentistry,
Kyungpook National University, Daegu 41940, Korea
8 Department of Biochemistry, School of Medicine, Jeju National University, Jeju 63243, Korea
* Correspondence: choiyh@deu.ac.kr; Tel.: +82-51-850-7413
Received: 2 July 2019; Accepted: 24 July 2019; Published: 26 July 2019
Abstract: Sea tangle (Laminaria japonica Aresch), a brown alga, has been used for many years as
a functional food ingredient in the Asia-Pacific region. In the present study, we investigated the
effects of fermented sea tangle extract (FST) on receptor activator of nuclear factor-κB (NF-κB) ligand
(RANKL)-stimulated osteoclast differentiation, using RAW 264.7 mouse macrophage cells. FST was
found to inhibit the RANKL-stimulated activation of tartrate-resistance acid phosphatase (TRAP) and
F-actin ring structure formation. FST also down-regulated the expression of osteoclast marker genes
like TRAP, matrix metalloproteinase-9, cathepsin K and osteoclast-associated receptor by blocking
RANKL-induced activation of NF-κB and expression of nuclear factor of activated T cells c1 (NFATc1),
a master transcription factor. In addition, FST significantly abolished RANKL-induced generation of
reactive oxygen species (ROS) by activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and
its transcriptional targets. Hence, it seems likely that FST may have anti-osteoclastogenic potential as
a result of its ability to inactivate the NF-κB-mediated NFATc1 signaling pathway and by reducing
ROS production through activation of the Nrf2 pathway. Although further studies are needed to
inquire its efficacy in vivo, FST appears to have potential use as an adjunctive or as a prophylactic
treatment for osteoclastic bone disease.
Keywords: fermented sea tangle; osteoclast differentiation; receptor of activator of nuclear factor
kappa-B ligand (RANKL); nuclear factor-κB (NF-κB); reactive oxygen species (ROS)
1. Introduction
Bone remodeling is an active physiological process involving bone deposition and bone resorption
by osteoblast and osteoclast, respectively. Imbalance of these processes in favor of resorption may
lead to the formation of osteolytic lesions and an increase in bone disease-related disorders and
morbidity [1–3]. Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) and macrophage
colony-stimulating factor (M-CSF) are cytokines that play important roles in osteoclast differentiation
and maturation. RANKL belongs to the tumor necrosis factor (TNF) superfamily and is regarded
as the key promoter of osteoclastogenesis. M-CSF by contrast, is involved in the maintenance of

Foods 2019, 8, 290
mature osteoclast survival and mobility [4,5]. The binding of RANKL to its receptor RANK results
in the activation of various signaling pathways, including the NF-κB pathway [6,7], which then
enhances the activation of nuclear factor of activated T cell c1 (NFATc1), which then in turn promotes
osteoclast formation by up-regulating the expression of osteoclast-specific genes [8,9]. In addition, a
number of previous studies have shown that reactive oxygen species (ROS) are also critical messengers
for osteoclast differentiation [10,11] and increased activity of the Nrf2 signaling system can block
this activation [12–14]. These findings suggest that suppression of ROS production in combination
with increasing activity of Nrf2 may provide a means to block osteoclast activity. Although various
drugs have been used clinically to inhibit bone resorption, all have severe side effects when used
long-term [15] and as a result, research into the prevention and treatment of osteolytic diseases using
natural products has greatly increased in recent years.
Many marine algae extract or components of these extracts have been shown to exhibit potential
for preventing and treating bone resorption related diseases [16,17] and fermented marine algal
extracts have attracted the attention of the food and medical care industries [17,18]. The sea tangle,
Laminaria japonica Aresch, is one of the most well-known edible brown seaweeds and has long been
used as an important food supplement in Pacific and Asian countries [19]. This seaweed is rich in
polysaccharides, dietary fiber, minerals, carbohydrates, polyphenols and proteins [20,21] and has
been reported to protect against obesity, inflammation and cancer [22–25]. Interestingly, Lee et al. [26]
developed a fermented form of sea tangle using Lactobacillus brevis with high antioxidant activity
and showed that a fermented sea tangle extract (FST) protected against liver damage better than a
non-fermented sea tangle extract [27,28]. They speculated that glutamate in the sea tangle which
converted to gamma-aminobutyric acid through the fermentation process, was the reason behind the
increased antioxidant capacity. It has been reported that FST supplementation reduce obesity and
improve stress management [29]. Furthermore, previous studies have shown that FST can protect
against age-associated short-term memory loss and reduced physical functioning [30–32]. However,
the effect of FST on bone has not previously been investigated and therefore we decided to investigate
whether FST had any inhibitory effect on RANKL-stimulated osteoclast differentiation using RAW
264.7 mouse macrophage cells.
2.1. Reagents and Antibodies

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and other reagents for cell
culture were purchased from WelGENE Inc. (Daegu, Republic of Korea). RANKL and osteoprotegerin
(OPG) were obtained from Abcam (Cambridge, MA, USA) and Peprotech (Rocky Hill, NJ, USA),
respectively. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tartrate-resistant
acid phosphatase (TRAP) assay kit, bovine serum albumin (BSA), 4 ,6-diamidino-2-phenylindole
(DAPI), 5,6-carboxy-2 ,7 -dichlorofluorescein diacetate (DCF-DA) and N-acetyl cysteine (NAC) were
purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). NE-PERTM nuclear and cytoplasmic
extraction reagents kit and polyvinylidene difluoride (PVDF) membranes were obtained from
Pierce Biotechnology (Rockford, IL, USA) and Schleicher & Schuell (Keene, NH, USA), respectively.
Fluorescein isothiocyanate (FITC)-phalloidin solution was purchased from Thermo Fisher Scientific
(Waltham, MA, USA). Primary and secondary antibodies were obtained from Santa Cruz Biotechnology
Inc. (Santa Cruz, CA, USA), Cell Signaling Technology Inc. (Beverly, MA, USA), Abcam, Novus
(Novus Biologicals, LLC., Littleton, CO, USA), Thermo Fisher Scientific and R&D system. Appropriate
horseradish-peroxidase (HRP)-linked secondary antibodies and enhanced chemiluminescence (ECL)
detection solution were purchased from Amersham Corp. (Arlington Heights, IL, USA). All reagents
not specifically identified were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
Foods 2019, 8, 290
2.2. Preparation of FST

FST received from Marine Bioprocess Co. Ltd. (Busan, Korea) was extracted as previously
described [30]. In brief, yeast extract and glucose were added to water at a ratio of 1:15 (w/v) and
sea tangle (L. japonica Aresch) was then added and sterilized in an autoclave at 121 ◦ C for 30 min.
After autoclaving, culture broth of L. brevis BJ20 (accession no. KCTC 11377BP) was added to the mix
at a concentration of 1.2% (v/v) and the mixture was incubated at 37 ◦ C for 2 days. The fermented
product was obtained by filtration and lyophilized. The dried extract (FST) so obtained was dissolved
in Milli-Q Water to produce a 10 mg/mL stock solution.
2.3. Cell Culture and Viability Analysis

RAW 264.7 cell line was purchased from the American Type Culture Collection (Manassas, VA,
USA). The cells were cultured in DMEM containing 10% heat inactivated FBS, penicillin (100 units/mL)
and streptomycin (100 g/mL) at 37 ◦ C in a humidified 5% CO2 atmosphere and subcultured every
3 days. The viability of the cells was assessed by MTT assay as previously described [14]. Briefly,
the cells were treated with the desired concentrations of FST with or without 100 ng/mL RANKL for
72 h and then incubated with 50 μg/mL MTT solution for 3 h. Formazan crystals were dissolved in
DMSO and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA)
microplate reader (Dynatech Laboratories, Chantilly, VA, USA) at 540 nm.
2.4. Osteoclast Differentiation and TRAP Assay

Osteoclast formation was measured by quantifying cells positively stained by TRAP. Briefly,
the cells were fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 10 min and then stained
with commercial TRAP staining kit according to the manufacturer’s instructions. Osteoclasts were
defined as TRAP-positive multinuclear cells containing 3 or more nuclei, under a phase-contrast
microscope (Carl Zeiss, Oberkochen, Germany). TRAP activity was determined in culture media using
a TRAP assay kit, in accordance with the manufacturer’s instructions. TRAP activities were expressed
as percentages of control activities.
2.5. F-Actin Ring Staining

As described previously, evaluation of actin ring formation was performed [14]. Briefly, the cells
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 5 min and then
stained with an anti-actin antibody at 4 ◦ C overnight. After washing with PBS, the cells were incubated
with FITC-conjugated phalloidin for 30 min at 37 ◦ C and then counterstained with 2.5 μg/mL DAPI for
20 min. F-actin rings were analyzed by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).
2.6. Western Blot Analysis

As described previously, total protein was extracted from the cells using the Bradford Protein
assay kit [14]. Nuclear and cytosolic proteins were prepared using a NE-PER nuclear and cytoplasmic
extraction reagents kit according to the manufacturer’s instructions. Equal amounts of protein from
samples were loaded and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat skim milk
in trisbuffered saline containing 0.1% Triton X-100 (TBST) for 1 h and probed with specific primary
antibodies at 4 ◦ C overnight (Table 1). After washing three times with TBST, the membranes were
incubated with the appropriate HRP-conjugated secondary antibodies for 2 h. Protein expression was
detected by an ECL kit and visualized by Fusion FX Image system (Vilber Lourmat, Torcy, France).
Foods 2019, 8, 290
Table 1. Information of primary and secondary antibodies.
Antibody Manufacturer Item No.

β-actin Santa Cruz sc-1615
CTSK Santa Cruz sc-48353
HO-1 Millipore 374090
IkBα Santa Cruz sc-371
Lamin B Santa Cruz sc-6216
MMP-9 Abcam 38898
NFATc1 Santa Cruz sc-7294
NF-κB p65 Santa Cruz sc-109
Phospho- NF-κB p65 Cell signaling 3033
Nrf2 Santa Cruz sc-13032
phospho-Nrf2 Abcam 76026
NQO-1 Novus NB200-209
OSCAR R&D system MAB1633
TRAP Thermo Fisher Scientific PA5-42729
Goat anti-mouse IgG-HRP Santa Cruz sc-2005
Goat anti-rabbit IgG-HRP Santa Cruz sc-2004
Bovine anti-goat IgG-HRP Santa Cruz sc-2350
Mouse anti-rabbit igG-TR Santa Cruz Sc-3917
CTSK: cathepsin K; HO-1: heme oxygenase-1; IκBα: inhibitory proteins of kappa B, alpha; MMP-9: matrix
metalloproteinase-9; NFATc1: nuclear factor of activated T cells c1; NF-κB: nuclear factor-kappa B; Nrf2: nuclear
factor-erythroid 2-related factor 2; NQO-1: NAD(P)H quinone oxidoreductase 1; OSCAR: osteoclast-associated
receptor; TRAP: tartrate-resistance acid phosphatase; HRP: horseradish-peroxidase.
2.7. Immunofluorescence Staining for NF-κB

RAW 264.7 cells were seeded on gelatin-coated glass coverslips. After it was cultured for 24 h,
cells were treated with RANKL in the presence or absence of various concentrations of FST for 24 h,
fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 15 min and
blocked with PBS containing 5% BSA. Cells were stained with primary antibody against phosphoNF-κ
B p65 at 4 ◦ C overnight and incubated with a fluorescein-conjugated anti-rat IgG in the dark at 37 ◦ C
for 1 h. Cells were mounted on slides and then analyzed by fluorescence microscope.
2.8. Measurement of Intracellular ROS Levels

The production of intracellular ROS was measured by a flow cytometer with DCF-DA as described
previously [14]. Briefly, the cells were treated with FST in the presence or absence of 100 ng/mL RANKL.
In the last 20 min of treatment, 10 μM DCF-DA was added to the incubated cells in the dark. Following
incubation, the cells were washed twice with PBS and 10,000 cells were analyzed for intracellular ROS
content by BD Accuri C6 software in a flow cytometer (BD Biosciences) at 480/520 nm. To observe ROS
generation by fluorescence microscopy, cells were stimulated with RANKL in the presence or absence
of FST for 1 h. Cells were then stained with DCF-DA and then fixed with 4% paraformaldehyde for 2.

All experiments were performed at least three times. Data were analyzed using GraphPad Prism
software (version 5.03; GraphPad Software, Inc., La Jolla, CA, USA) and expressed as the mean
± standard deviation (SD). Differences between groups were assessed using analysis of variance
followed by ANOVA-Tukey’s post hoc test and p < 0.05 was considered to indicate a statistically
significant difference.
Foods 2019, 8, 290
3. Results
3.1. Effect of FST on Cell Viability in RAW 264.7 Cells

RAW 264.7 cells were treated with various concentrations of FST for 72 h and then 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. Figure 1A shows
that FST had no cytotoxicity on the cells at concentrations up to 800 μg/mL but relatively cytotoxic
effect was observed in the 1000 μg/mL treatment group as compared with untreated controls. In the
presence of 100 ng/mL RANKL or 100 ng/mL osteoprotegerin (OPG), a decoy receptor for RANKL that
inhibits osteoclastogenesis [33,34], cell viability was not significantly reduced by FST at concentrations
up to 800 ng/mL compared to that of control groups (Figure 1B). Hence, non-toxic concentrations
(<800 μg/mL) were used to investigate the effect of FST on RANKL-induced osteoclast differentiation.
Figure 1. Effects of fermented sea tangle extract (FST) and receptor of activator of nuclear factor
kappa-B ligand (RANKL) on the viability of RAW 264.7 mouse macrophage-like cells. Cells were
treated with desired concentrations of FST in the absence (A) or presence (B) of 100 ng/mL RANKL
and/or 100 ng/mL OPG for 72 h. H2 O2 was used as a positive control. Cell viabilities were measured by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Relative cell viability is
expressed as percentages compared to treatment of naïve control cells. Results are presented as means
± SD of three independent experiments. * p < 0.05 and *** p < 0.005 indicates significant difference
compared to the untreated control cells. OPG: osteoprotegerin; +: cells treated the reagent; -: cells
untreated the reagent.
3.2. FST Suppresses RANKL-Induced Osteoclastogenesis in RAW 264.7 Cells

In order to examine the effect of FST on RANKL-induced osteoclastogenesis, RAW 264.7 cells
were treated with FST in the presence of different concentrations of RANKL. As shown in Figure 2A,
FST treatment markedly inhibited RANKL-induced osteoclast-like morphological changes. TRAP
staining demonstrated that FST suppressed cell fusion and the conversion of RAW 264.7 cells into
osteoclasts (Figure 2B FST suppressed numbers of TRAP-positive osteoclasts as compared with RANKL
treated cells, dose-dependent manner (Figure 2C). These reductions in TRAP-positive osteoclast
number were paralleled by the inhibition of TRAP activity (Figure 2D). As expected, RANKL-induced
osteoclast differentiation and TRAP activity were completely suppressed in the presence of OPG.
3.3. FST Disrupts RANKL-Induced Formation of F-Actin Rich Adhesive Structures in RAW 264.7 Mouse
Macrophage-Like Cells
Formation of the F-actin rich adhesive structures by osteoclasts is an essential step in bone
resorption [35,36]. Figure 3 indicated that staining with FITC-conjugated phalloidin showed
RANKL (100 ng/mL) stimulation increased well-defined F-actin sealing rings with a higher intensity
ring height. However, the size of rings formed by RANKL-treated cells was remarkably and
concentration-dependently reduced in cells co-treated with FST. Furthermore, OPG treatment
complementally inhibited the F-actin sealing ring formation in RANKL-stimulated cells.
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Figure 2. Inhibition of RANKL-stimulated osteoclast differentiation by FST in RAW 264.7 mouse

macrophage-like cells. Cells were stimulated with 100 ng/mL RANKL in the presence or absence of
FST or 100 ng/mL OPG for 5 days. (A) Representative photographs of the morphological changes are
presented. (B) Cells were fixed and stained for TRAP and examined under an inverted microscope.
(C) TRAP-positive multinucleated cells were counted to determine osteoclast numbers. (D) Supernatants
were collected from cells grown under the same conditions and TRAP activities were measured using
an ELISA reader. Results are presented as means ± SD of three independent experiments. * p < 0.05,
** p < 0.01 and *** p < 0.001 indicates significant difference compared to RANKL-treated cells. RANKL:
receptor of activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract; OPG:
osteoprotegerin; TRAP: tartrate-resistance acid phosphatase; +: cells treated the reagent; -: cells
untreated the reagent.
Figure 3. Suppression of F-actin ring formation by FST in RANKL-induced RAW 264.7 mouse
macrophage-like cells. The cells were co-treated with 100 ng/mL RANKL in the presence or absence
of FST or 100 ng/mL OPG for 5 days and stained for F-actin rich adhesive structures with fluorescein
isothiocyanate (FITC)-phalloidin and 4 ,6-diamidino-2-phenylindole (DAPI). The photographs are
representative of the morphological changes observed under a fluorescence microscope. RANKL:
receptor of activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract.
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3.4. FST Inhibits the RANKL-Induced Nuclear Translocation of NF-κB and IκBα Degradation in RAW
264.7 Cells
Activation of NF-κB through nuclear translocation by RANKL is an essential step for initiation of
osteoclast differentiation [6,7]. Therefore, we assessed whether FST affected the activation of NF-κB
induced by RANKL. As shown in Figure 4A,B, our immunoblotting results reveal that the expression
of NF-κB was markedly increased in the nuclei of RANKL treated cells but the expression of IκBα was
reduced in the cytoplasm, which suggested that RANKL stimulated activation of NF-κB. However,
FST suppressed the RANKL-mediated degradation of IκBα and the subsequent nuclear accumulation
of NF-κB. Furthermore, immunofluorescence studies produced similar results. More specifically,
phosphorylated NF-κB p65 was predominantly located in nuclei in RANKL-stimulated cells but not in
FST and RANKL co-treated cells (Figure 4C).
Figure 4. Effects of FST on the RANKL-induced activation of NF-κB in RAW 264.7 mouse
macrophage-like cells. (A) After co-treating cells with 100 ng/mL RANKL in the presence or absence of
FST for 1 h, nuclear and cytosolic proteins were isolated. The expression of NF-κB and IκB-α were
determined by Western blotting. Lamin B and β-actin were used as internal controls for the nuclear
and cytosolic fractions, respectively. (B) Densitometry quantifications of protein expressions were
measured by ImageJ. Statistical analyses were conducted using analysis of variances between groups.
*** p < 0.0001 when compared to control. (C) Cells grown on gelatin-coated glass coverslips were
co-treated with 800 μg/mL FST with or without 100 ng/mL RANKL. Localization of phospho-NF-κB
p65 was observed under a fluorescence microscope following staining with anti-phospho-NF-κB p65
antibody (red) and DAPI (nuclear stain; blue). Original magnification ×400. RANKL: receptor of
activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract; IκBα: inhibitory proteins
of kappa B, alpha; NF-κB: nuclear factor-kappa B; +: cells treated the reagent; -: cells untreated
the reagent.
3.5. FST Down-Regulates RANKL-Induced Osteoclast-Associated Gene Expression in RAW 264.7 Cells
NFATc1 is considered to be the most important regulator of the transcriptional activation
of osteoclast differentiation-associated genes by RANKL [8,9]. To examine in more detail the
mechanism of FST-mediated inhibition of osteoclastogenesis, we assessed the expression of NFATc1 in
RANKL-stimulated RAW 264.7 cells. Consistent with previous studies, the expression of NFATc1 was
significantly increased by RANKL but was down-regulated in a concentration-dependent manner by FST
(Figure 5). In addition, we investigated the effects of FST on the levels of specific marker for osteoclast
such as TRAP, cathepsin (CTSK), matrix metallopeptidase-9 (MMP-9) and osteoclast-associated receptor
Foods 2019, 8, 290
(OSCAR). Figure 5 showed that RANKL markedly up-regulated levels of these osteoclast-specific
markers, which were effectively attenuated by the addition of FST. Co-treatment with OPG also
completely prevented increases in these protein markers.
Figure 5. Inhibition of the RANKL-induced expressions of osteoclast-regulatory genes by FST in RAW

264.7 mouse macrophage-like cells. Cells were co-treated with various concentrations of FST or 100
ng/mL OPG in the presence or absence of 100 ng/mL RANKL for 5 days. (A) The expression levels of
osteoclast-regulatory proteins were assessed by Western blot analysis. β-actin was used as the internal
control. The results shown are representative of three independent experiments. (B) Densitometry
quantifications of protein expression were measured by ImageJ. Statistical analyses were conducted
using analysis of variances. * p < 0.05 and *** p < 0.0001 when compared to control. ### p < 0.0001
when compared to RANKL treatment. RANKL: receptor of activator of nuclear factor kappa-B
ligand; FST: fermented sea tangle extract; OPG: osteoprotegerin; CTSK: cathepsin K; MMP-9: matrix
metalloproteinase-9; NFATc1: nuclear factor of activated T cells c1; OSCAR: osteoclast-associated
receptor; +: cells treated the reagent; -: cells untreated the reagent.
3.6. FST Attenuates RANKL-Induced Intracellular ROS Accumulation Associated with Activation of Nrf2 in
RAW 264.7 Mouse Macrophage-Like Cells
Overproduction of intracellular ROS plays a critical step in RANKL-mediated
osteoclastogenesis [12–14], thereby we examined whether FST inhibits the generation of ROS during
RANKL-mediated osteoclastogenesis using DCF-DA, a cell permeant redox-sensitive dye. We
demonstrated by flow cytometry that ROS levels were significantly increased by RANKL and that
these up-regulation were abolished by FST (Figure 6A,D). Moreover, this effect of FST was supported
by our fluorescence microscopic examination (Figure 6B) and further, co-treatment with N-acetyl
cysteine (NAC), an intensive ROS scavenger, completely alleviated RANKL-induced ROS generation
and F-actin ring formation (Figure 6C). In addition, Figure 6E,F shows that FST has the efficacy of
equivalence and/or superiority compared with NAC and it was suggested that FST is a powerful
anti-oxidant, thereby it has a suppressed RANKL-mediated ROS generation.
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Figure 6. Effect of FST on RANKL-induced reactive oxygen species (ROS) generation in RAW
264.7 mouse macrophage-like cells. Cells were co-treated with 100 ng/mL RANKL for 1 h in the
presence or absence of 800 μg/mL FST or 10 mM NAC. (A,D) Cells were stained with 5,6-carboxy-2 ,
7 -dichlorofluorescein diacetate (DCF-DA) and DCF fluorescence was measured by flow cytometry.
Results are means of two independent experiments. (B) After staining with DCF-DA, images were
obtained using a fluorescence microscope. Images are representative of at least three independent
experiments. (C) Cells cultured under the conditions used to induce osteoclast differentiation were fixed
and stained for F-actin ring with FITC-phalloidin solution and imaged under a fluorescence microscope.
Representative photographs of the morphological changes observed are presented. (E) Cellular proteins
were isolated from cells and the expression of NFATc1, phospho-NF-κB and phosphor-Nrf2 by Western
blot analysis. β-actin was used as the internal control. The results shown are representative of three
independent experiments. (F) Statistical analyses were conducted using analysis of variances. * p < 0.05
and *** p < 0.001 when compared to control. ### p < 0.0001 when compared to RANKL treatment.
RANKL: receptor of activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract;
NAC: N-acetyl cysteine osteoprotegerin; NFATc1: nuclear factor of activated T cells c1; p- NF-κB p65:
phosphorylated nuclear factor-kappa B p65; p-Nrf2: phosphorylated nuclear factor-erythroid 2-related
factor 2; +: cells treated the reagent; -: cells untreated the reagent.
Foods 2019, 8, 290
In addition, we show that FST increased the expression and phosphorylation of Nrf2
in RANKL-stimulated cells, which was associated with an increase in typical Nrf2-dependent
cytoprotective enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H: Quinone oxidoreductase 1
(NQO-1) (Figure 7A,B). Furthermore, we observed that Nrf2 translocation to the nucleus was promoted
by FST treatment (Figure 7C,D).
Figure 7. Activation of Nrf2 signaling pathway by FST in RAW 264.7 mouse macrophage-like cells.
Cells were treated with FST with or without 100 ng/mL RANKL for 5 days. (A) Total cellular proteins
were isolated from cells and the expression levels of Nrf2 and its regulatory proteins were assessed
by Western blot analysis. β-actin was used as the internal control. (C) The expression of nuclear and
cytosol Nrf2 were determined by Western blotting. Lamin B and β-actin were used as internal controls
for the nuclear and cytosolic fractions, respectively. The results shown are representative of three
independent experiments. (B,D) Statistical analyses were conducted using analysis of variances between
groups. * p < 0.05 and *** p < 0.0001 when compared to control. # p < 0.05 and ### p < 0.0001 when
compared to RANKL treatment. RANKL: receptor of activator of nuclear factor kappa-B ligand; FST:
fermented sea tangle extract; Nrf2: nuclear factor-erythroid 2-related factor 2; p-Nrf2: phosphorylated
nuclear factor-erythroid 2-related factor 2; HO-1: heme oxygenase-1; NQO-1: NAD(P)H quinone
oxidoreductase 1; +: cells treated the reagent; -: cells untreated the reagent.
4. Discussion
Osteoclasts are multinucleated cells of hematopoietic origin which are derived from the
monocyte/macrophage in their ability to resorb bone, whereas osteoblast are derived from pluripotent
mesenchymal stem cells and are involved in bone formation [1,2]. Since excessive bone resorption
by osteoclasts causes an imbalance in bone regeneration and induces osteolytic diseases, osteoclasts
are considered prime targets for the management and treatment of bone diseases [2,3]. RANKL is a
pro-osteoclastogenic cytokine and plays a crucial role in promoting osteoclastogenesis from osteoclast
progenitor cells [4,5]. As has been well established in many earlier studies, RANKL binds to RANK
expressed on the plasma membrane of osteoclast precursors and activates complex signaling cascades
including NF-κB and NFATc1 for osteoclast differentiation [9,10]. Differentiation through activation
of these signal transduction systems by RANKL is characterized by the formation of multinucleated
giant cells [5,7]. This is a preliminary step in the maintenance, formation and function of the F-actin
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loop structure, which plays an important role in seal zone formation and resorption of bone mineral
matrix in osteoclasts by activated TRAP [37,38]. According to the present findings, FST effectively
inhibited RANKL-induced TRAP activation and F-actin ring formation without causing any significant
cytotoxicity in RAW 264.7 cells, implying that FST suppressed osteoclast differentiation from osteoclast
precursors at an early stage.
NF-κB, a transcription factor that plays a key role in inducing osteoclast differentiation, complexes
with cytoplasmic IκB-α in the absence of osteoclastogenic induction signals and keeps it in an inactive
form that tightly regulates its transcriptional activity for osteoclast differentiation [4,37]. However,
the interaction of RANKL and RANK promotes the activation of the IκB kinase (IKK) complex,
which phosphorylates IκB-α leading to ubiquitin-dependent degradation [6,7]. As a result, free NF-κB
translocates to the nucleus and activates transcription of various genes involved in osteoclastogenesis [5].
Our results demonstrated that RANKL promoted the degradation of IκB-α in the cytoplasm and
induced the translocation of NF-κB into the nucleus, both of which are essential for the activation of
NF-κB but that these changes were completely inhibited by FST.
In the early stages of NF-κB activation and osteoclast differentiation, NFATc1 acts as a master
regulator that enhances transcription of various osteoclast marker genes, which are highly expressed
in the terminal differentiation stage to promote bone resorption [5,39]. In addition to blocking
RANKL-induced NF-κB activation, FST inhibited NFATc1 expression in RANKL-treated cells.
OPG treatment also completely blocked the expression of NFATc1. As further proof that FST was
effectively inhibiting osteoclastogenesis, we showed that it attenuated RANKL-induced up-regulation
of osteoclast marker genes such as TRAP, MMP-9, CTSK and OSCAR to levels seen in the control and
OPG co-treated groups. Although further experiments are required to determine whether NFATc1
inhibition is the direct result of NF-κB inactivation, the present results indicate that inactivation of the
NF-κB signaling pathway and inhibition of the expression of osteoclast marker genes associated with a
decrease in NFATc1 expression are involved as important mechanisms in the anti-osteoclastogenic
effect of FST.
A number of previous studies have shown that ROS, as specific secondary messengers, play a
key role in the initiation of RANKL-stimulated osteoclast differentiation and bone resorption through
similar pathways involving the activation of NF-κB and NFATc1 [10,12]. However, the accumulation of
excessive ROS due to oxidative stress blocks osteoblast differentiation, suppresses osteoblast survival
and acts to promote bone loss [12,13]. It has also been reported that a variety of natural products with
antioxidant activity inhibit osteoclast differentiation by inhibiting ROS production [6,10,11]. Therefore,
ROS can be considered a potential target for inhibition of osteoclast differentiation and prevention
of bone loss. The present results showed that FST significantly suppressed ROS production by
RANKL. Moreover, consistent with the results of previous studies [12,40], RANKL-induced osteoclast
differentiation was completely inhibited when production of ROS was artificially blocked using NAC,
indicating that FST blocks osteoclast differentiation by acting as a scavenger or inhibitor of ROS. In order
to reduce the damage from oxidative stress in the face of excess production of ROS in cells, several
transcription factors are known to be activated to increase the expression of downstream antioxidant
enzymes [41,42]. One of these redox sensitive transcription factors, Nrf2 has recently been reported to
attenuate osteoclast differentiation through the regulation of ROS production [42,43]. For example,
Nrf2 deficiency improved RANKL-induced osteoclast differentiation [44], whereas local induction of
nuclear Nrf2 weakened RANKL-mediated osteoclastogenesis [45]. Under normal conditions, Nrf2 is
sequestered by Kelch-like ECH-associated protein 1 (Keap1) to the cytoplasm but becomes separated
from Keap1 by oxidative or electrophilic stress and translocated into the nucleus. In the nucleus,
Nrf2 binds to the antioxidant response elements to induce the transcription of target antioxidants and
detoxifying enzymes including HO-1 and NQO-1 [42,43]. In this study, FST significantly increased
expression of Nrf2 and its transcriptional targets, including HO-1 and NQO-1 in RANKL-treated RAW
264.7 cells. We also observed that FST increased phosphorylation and nuclear translocation of Nrf2
compared to the RANKL-alone stimulated group. The results presented, indicate that FST attenuates
Foods 2019, 8, 290
osteoclast differentiation by decreasing RANKL-induced oxidative stress in osteoclast precursor cells

through the activation of Nrf2 and its downstream genes.
5. Conclusions
To assume the effect of FST on RANKL-mediated osteoclast differentiation, recombinant RANKL
protein was used to differentiate murine monocyte/macrophage RAW 264.7 cells as osteoclast
precursor cells into osteoclasts. Present results demonstrated that FST inhibited RANKL-induced
osteoclastogenesis and reduced the expression of several key osteoclast-regulatory genes through the
inactivation of NF-κB. In addition, FST blocked RANKL-induced oxidative stress, which was associated
with the activation of Nrf2 signaling pathway. Although the present study provides new insights
into the inhibition of osteoclastogenesis by FST, further investigation of the molecular mechanisms
underlying this process as well as identification of the bioactive constituents of FST are needed.
Author Contributions: Y.H.C., Y.-J.J., C.P. and B.-J.L. conceived and designed the experiments; J.-W.J., H.L.,
S.H.H. and C.P. performed the experiments; S.Y.J., H.L., G.-Y.K., E.K.P. and J.W.H. analyzed the data; J.-W.J. wrote
the paper and Y.H.C. edited the paper.
Funding: This research was a part of the project titled ‘Development of functional food products with natural
materials derived from marine resources (20170285)’, funded by the Ministry of Oceans and Fisheries.
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foods
Article
Antioxidant Activities of Solanum nigrum L. Leaf
Extracts Determined in In Vitro Cellular Models
Agata Campisi 1 , Rosaria Acquaviva 1 , Giuseppina Raciti 1 , Anna Duro 2 , Milena Rizzo 1, * and
Natale Alfredo Santagati 1
1 Department of Drug Science, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy;
agcampisi@gmail.com (A.C.); racquavi@unict.it (R.A.); racitigi@unict.it (G.R.); santagat@unict.it (N.A.S.)
2 Department of Biological, Geological and Environmental Sciences, University of Catania, Via A. Longo 19,
95125 Catania, Italy; annaduro@unict.it
* Correspondence: milena.rizzo@unict.it
Received: 31 December 2018; Accepted: 30 January 2019; Published: 8 February 2019
Abstract: Several medicinal foods abound in traditional medicine with antioxidant potentials that
could be of importance for the management of several diseases but with little or no scientific
justification to substantiate their use. Thus, the objective of this study was the assessment of
the antioxidant effect of two leave extracts of Solanum nigrum L. (SN), which is a medicinal plant
member of the Solanaceae family, mainly used for soup preparation in different parts of the world.
Then methanolic/water (80:20) (SN1) and water (SN2) leaves extracts were prepared. The total
polyphenolic content and the concentration of phenolic acids and flavones compounds were
determined. In order to verify whether examined extracts were able to restore the oxidative status,
modified by glutamate in primary cultures of astrocytes, the study evaluated the glutathione levels,
the intracellular oxidative stress, and the cytotoxicity of SN1 and SN2 extracts. Both extracts were
able to quench the radical in an in vitro free cellular system and restore the oxidative status in in vitro
primary cultures of rat astroglial cells exposed to glutamate. These extracts prevented the increase in
glutamate uptake and inhibited glutamate excitotoxicity, which leads to cell damage and shows a
notable antioxidant property.
Keywords: Solanum nigrum L. leave extracts; natural products; antioxidant activity; functional food
1. Introduction
Several medicinal foods abound in traditional medicine with antioxidant activities. This could
be of important for the management of several diseases but has little or no scientific justification to
substantiate their use. In the tropics, as in Asia and in sub-Saharan Africa, green leafy vegetables are
used as one of the major components of local dishes.
Solanum nigrum L. (SN) belongs to the Solanaceae family to Europe, Asia, and North America and
was introduced in South America, Australia, and Africa. It represents one of the largest and most
variable species groups of the genus. SN, well known as “Black Nightshade” (the English name), is an
herbal plant widely distributed throughout the world, extending from tropical regions to temperate
regions [1].
In many developing countries, SN constitutes a minor food crop, with the shoots and berries not
only used as vegetables and fruits but also for various medicinal and local uses [2]. SN serves mainly
as vegetables for soup preparation in different parts of the world. Several studies have investigated
the nutritive value of the ‘vegetable black nightshade,’ which put forward evidence that this species
constitutes a nutritious vegetable [3]. This plant was chosen not only for being nutritive, but also
for their folkloric reports of medicinal properties [4]. Studies document potential health benefits of
different parts of this vegetable. SN leaves have been reportedly used in traditional medicine for the

Foods 2019, 8, 63
management of several diseases including seizure and epilepsy, pain, ulcer, inflammation, diarrhea,
some eye infections, and jaundice [5,6].
In folklore medicine, the leaves are used to treat oral ulcers in India where an interesting
pharmacological investigation has been performed by using an aqueous extract of SN leaves [7].
More recently, many research studies have reported that SN showed anti-cancer activity for
hepatocellular carcinoma cells [8], human ovarian carcinoma cells [9], human colorectal carcinoma
cells [10], and human endometrial carcinoma cells [11]. The leaves can provide appreciable amounts of
protein and amino acids, minerals including calcium, iron, and phosphorus, vitamins A and C, fats and
fibers, and appreciable amounts of methionine, which is an amino acid scarce in other vegetables. Other
chemical constituents reported in leaves are steroidal glycosides [12]. Very recently, from the unripe
berries, a previously undescribed steroidal alkaloids [13] and steroidal glycosides [14] were isolated.
Those compounds showed a potent inhibitory activity against the lipopolysaccharide (LPS)-induced
nitric oxide (NO) production.
Because medicinal plants are gaining popularity for the production of reliable and safe medicines
suitable for human, many studies investigated the composition of extracts, their biological activities,
and optimization of extraction procedures [15,16].
The extracts of the SN contain many polyphenolic compounds. The leaves are rich in polyphenols,
including phenolic acids and flavones [17]. Zaidi et al. demonstrated that treatment of rats with SN
leaves extract was able to reduce oxidative stress, and, in particular, they showed the potential of
this extract in preventing/alleviating stress-induced diseases, involving oxidative damage to cellular
constituents especially the brain [18]. Antioxidant activity might be due to the presence of the
above-mentioned polyphenolic compounds on SN stems and leaves [19]. Sun et al. demonstrated that
oxidative stress has been associated with pathological conditions, including Central Nervous System
(CNS) diseases and physiological brain aging processes [20].
A very interesting study has shown that dietary inclusions of Solanum leaf could protect against
cognitive and neurochemical impairments induced by scopolamine, and, hence, this vegetable could
be used as a source of functional foods and nutraceuticals for the prevention and management of
cognitive impairment-associated diseases such as Alzheimer’s disease [21].
The formation and release of Radical Oxygen reactive Species (ROS) cause structural and
functional alterations of cell membranes. Free radicals attack polyunsaturated fatty acids in
bio-membranes and mitochondria begin the main source of ROS, when the mitochondrial respiratory
chain is impaired. In these cases, a compound possessing antioxidant properties can be useful in
stopping ROS production and limiting oxidative cell damages, which is particularly interesting if
this activity is produced by a functional food [22]. Experimentally, ROS is well determined by using
reduced glutathione (GSH), which is known as the most important scavengers of reactive species, and
a reduced glutathione/oxidized glutathione ratio is used as a marker of oxidative stress [23].
At the central level, the oxidative stress may activate several calcium-dependent enzymes, causing
mitochondria impairment, a decrease in adenosine triphosphate (ATP) levels, ROS production,
and subsequent neuronal cell death [24]. A brief exposure to glutamate, which is a major
excitatory neurotransmitter in the CNS, could determine several acute and chronic brain damages
on differentiated astrocytes, which then causes cell swelling, whereas a prolonged incubation
(excitotoxicity) induces cell damage [25,26]. This phenomenon causes alterations in glutamate
transport, GSH depletion, and macromolecular synthesis [27].
Herein, we prepared two SN polar leaf extracts and assessed the total polyphenolic content and
the concentration of phenolic acids and flavones compounds. Antioxidant activity in both an in vitro
cellular free system and in vitro cellular system was evaluated. To verify whether SN1 and SN2 extracts
were able to restore the oxidative status, which were modified by glutamate in primary cultures of
astrocytes, GSH, ROS levels and the cytotoxicity of both extracts has been assessed.
Foods 2019, 8, 63
2.1. Materials
Reference compounds (gallic acid, protocatechuic acid, chlorogenic acid, gentisic acid, caffeic
acid, luteolin, apigenin) were purchased from Sigma (St. Louis, MO, USA). Acetonitrile,
methanol, and water were chromatographic-grade and were bought from Carlo Erba (Milano,
Italy). STable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-
tetrazolium bromide (MTT), 2 ,7 -dichlorofluorescein diacetate (DCFH-DA), 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox), dimethylsulfoxide (DMSO), GYKI 52466, phosphate
buffer solution (PBS), and other analytical chemicals were purchased from Sigma-Aldrich Chimica
(Milan, Italy). Distilled and deionized water was used for the preparation of all samples and solutions
and they were used after filtration through HA filters (0.45 μm Millipore, Bedford, MA, USA). All
standards were diluted to the appropriate obtained concentration and were stored at +4 ◦ C in amber
vials in a dark place until analysis. Dulbecco s modified Eagle s medium (DMEM) and heat-inactivated
Fetal Bovine Serum (FBS) were obtained from Invitrogen (Milan, Italy). The mouse monoclonal
antibody against glial fibrillary acidic protein (GFAP) and anti-IgG polyclonal antibody were from
Chemicon (Prodotti Gianni, Milan, Italy).
2.2. Methods
2.2.1. Plant Material and Extraction Procedures

SN leaves were collected around Catania (Sicily, Italy) in June 2018 and air-dried at room
temperature (24 ± 2 ◦ C) with no direct light. A voucher specimen of the plant has been deposited
(N. 8234) in the herbarium of the Botany Department of the University of Catania (Italy). The dried and
powdered leaves (5 g) were extracted by maceration for 24 h at room temperature with 150 mL each
of methanol/water solution 80:20 v/v (SN1) and water (SN2), respectively. The extraction procedure
was repeated three times, and the solvent extracts were combined and separated from the residue by
filtration through Whatman N. 1 filter paper in a Buchner funnel under vacuum. The methanol was
removed under a reduced pressure below 40 ◦ C by using a rotary evaporator, and the aqueous phase
remaining after evaporation of the organic phase was freeze-dried. The water extract was freeze-dried.
The obtained dried extracts were 1.305 ± 0.24 g for SN1 and 0.975 ± 0.31 g for SN2, respectively.
The obtained dried extracts were stored at −20 ◦ C until undergoing assays.
2.2.2. Determination of Total Phenolic Content

The amount of total phenolic in the studied extracts was determined using a modified
Folin-Ciocalteu method [28]. Extracts were prepared at a concentration of 0.5 mg/mL. An aliquot of
a known dilution of the extract was mixed with 5 mL Folin–Ciocalteu reagent (previously diluted
10-fold with deionized water) and was allowed to react for 6 min. Then, 4 mL (70 g/L) of sodium
carbonate solution was added to test tubes. The tubes were vortexed for 20 s and allowed to stand
for 90 min for color development. Absorbance was measured at 760 nm by using the Perkin Elmer
UV–Vis Lambda 25 spectrophotometer (Perkin Elmer Italia spa, Monza, Italy). Extract samples were
evaluated at the final concentration of 1 mg/mL. The measurements were compared to a standard
curve of prepared gallic acid solutions. The total phenolic content were 92.2 mg/g of extract for SN1
and 40.0 mg/g of extract for SN2, which was expressed as a gallic acid equivalent.
2.2.3. Chromatography
Chromatographic analysis was performed by using high-performance liquid chromatography
(HPLC) (Perkin Elmer, Norwalk, CT, USA) Series 200 pump equipped with an LC-235C Diode Array
Detector (DAD), auto-sampler, and column oven. Chromatographic data were processed by using a
Turbochrom Workstation software, version 6.1.2 (Perkin Elmer, Norwalk, CT, USA). Separation and
Foods 2019, 8, 63
determinations were accomplished on a 5 μm Hypersil ODS RP-18 column (250 × 4.6 mm, Supelco,
Bellefonte, PA, USA) fitted with a guard column (Hypersil ODS RP-18, 5 μm particles, 10 × 4.6 mm,
Supelco, Bellefonte, PA, USA). All the samples were filtered, through 0.45 μm membrane filter, and
degassed by an ultrasonic bath before the injection. The procedure was performed, as previously
described [17].
2.2.4. Determination of Antioxidant Activity in an In Vitro Cellular Free System

The stable DPPH radical was used for the determination of free radical-scavenging activity of the
extracts [29]. Because of its odd electron, the DPPH radical gives a strong absorption band at 517 nm
in visible spectroscopy (deep violet color). As this electron becomes paired off in the presence of a
free radical scavenger, the absorption vanishes, and the resulting decolorizing is stoichiometric with
respect to the number of electrons taken up. The reaction mixture contained 86 μM DPPH radical and
different concentrations of each extracts (0.025-0.5-0.1-0.2-0.4 mg/mL) in 1 mL of ethanol. After 10 min
at room temperature, the absorbance at 517 nm was recorded [30]. Trolox (30 μM), water-soluble
derivative of vitamin E, was used as a standard. The assay was performed in triplicates.
2.2.5. Determination of Antioxidant Activity within an In Vitro Cellular System

Primary cultures of astrocytes were prepared from new-born albino rat brains (from 1-day-old to
2-day-old Wistar strain rats) as described [31]. Cerebral tissues, after dissection and careful removal
of the meninges, were mechanically dissociated through 82 μm pore sterile mesh (Nitex, Darmastadt,
Germany). Isolated cells were suspended in DMEM, supplemented with 20% (v/v) FBS, 2 mM glutamine,
streptomycin (50 mg/mL), and penicillin (50 U/mL), and plated at a density of 3 × 106 cells/100 mm
dishes and of 0.5 × 105 cells/chamber of multi-chambered slides. Cells were maintained at 37 ◦ C in a
5% CO2 and 95% air humidified atmosphere for two weeks. The medium was exchanged every three
days. The low initial plating density of dissociated cells was meant to favor the growth of astrocytes
with only a very little oligodendroglial and microglial cells contamination. Astroglial cell cultures were
characterized at 14 days in vitro (DIV), when it is confluent, by immunofluorescence staining with GFAP.
All experiments conformed to the guidelines of the local Ethical Committee (University of Catania, Italy),
and were carried out in accordance with the EC Directive 86/609/EEC for animal experiments.
Astrocytes at 14 DIV were treated with glutamate (500 μM) for 24 h, as previously described [32].
Those cultures were treated with different concentrations of SN1 and SN2 (0.5 and 1 mg/mL), which
were added 20 min before glutamate exposure. Four replicates were carried out for each sample. In a
subset of experiments, to assess the inhibition of glutamate effects, the astroglial cell cultures were
incubated 20 min prior to glutamate exposure, with GYKI 52466 (100 μM), the specific AMPA/KA
receptor antagonist.
Astroglial cell survival analysis was performed by the MTT reduction assay, which evaluated
mitochondrial dehydrogenase activity, as previous reported [33,34]. Astrocytes were set up 0.5 × 105
cells per well of a 96-multiwell, flat-bottomed, 200-μL micro plate. They were maintained at 37 ◦ C in
a humidified 5% CO2 and 95% air mixture [33]. At the end of treatment time, 20 μL of 0.5% MTT in
(pH 7.4) PBS were added to each micro-well. After 1 h of incubation with the reagent, the supernatant
was removed and replaced with of DMSO (200 μL). The optical density of each well was measured
with a micro-plate spectrophotometer reader (Titertek Multiskan, Flow Laboratories, Helsinki, Finland)
at l = 570 nm.
2.2.6. Glutathione Measurement

Astroglial cell cultures were scraped off and lysed in 50 μM sodium phosphate buffer (pH 7.4).
The Bradford assay determined the protein concentration in cell extracts [32]. Then, a Hitachi U-2000
spectrophotometer (Hitachi, Tokyo, Japan) chemically determined the total glutathione intracellular
content (GSH + GSSG), as described by Chen YH et al. [35].
Foods 2019, 8, 63
2.2.7. ROS Levels Determination

Reactive species determination was performed by using DCFH-DA as a fluorescent probe.
Furthermore, 100 μM of DCHF-DA was dissolved in 100% methanol, added to the cellular medium,
and the cells were incubated at 37 ◦ C for 30 min. Under these conditions, the acetate group was not
hydrolyzed [12]. After incubation, astroglial cell cultures were lysed and centrifuged at 10,000× g for
10 min. The fluorescence corresponding to the radical oxidized species 2 ,7 -dichlorofluorescein (DCF)
was monitored by measuring the excitation (λ = 488 nm) and emission (λ = 525 nm), using an F-2000
spectrofluorometer (Hitachi).
Values are expressed as a percentage of fluorescence intensity per mg protein versus control
(% I.F/mg prot vs. control). Protein concentration was measured, according to the Bradford assay
applied by Li Volti et al [32].

All values are presented as means ± S.D. of five separate measurements. Statistical analysis
was performed using one-way ANOVA, which was followed by the Newman-Keuls post-hoc test.
Differences were considered statistically significant at * p < 0.05 and ** p < 0.001.
3. Results
3.1. Analysis of SN Extracts

The total phenolic contents was assayed by a modified Folin-Ciocalteu method using gallic acid,
and is reported in Table 1, with the physical characteristics and the dry weight yields of SN1 and SN2.
Table 1. Extraction yields and concentration of total phenolic content of SN1 and SN2 extracts (n = 3).
Total Phenolic Content

Extract Solvent Physical Yield %
(mg/g of Extract)
MeOH-H2 O
SN1 Brown solid 26.1 ± 3.9% 92.2 ± 4.8
80–20 v/v
SN2 H2 O Brown solid 19.5 ± 5.0% 40.0 ± 6.9
The relative concentrations of phenolic acids and flavones in SN1 and SN2 were determined by
HPLC analysis, according to Huang et al. [29]. The values were expressed in mg/g of dry extract.
The results of the five phenolic acids determination (gallic, protocatechuic, chlorogenic, gentisic, and
caffeic) and two flavones (luteolin and apigenin) in studied extracts are summarized in Table 2 while
Figure 1 shows a representative chromatogram.
It was found that the major compound in both extracts was chlorogenic acid, whereas
gentisic acid is the second. Luteolin is more abundant than apigenin, and less amounts of caffeic
acid and protocatechuic acid were found. Gallic acid exists only in traces together with other
unknown compounds.
Table 2. Contents of phenolic acid and flavones (mg/g of dry weight) in SN1 and SN2 leave extracts
(n = 3).
Gallic Protocatechuic Chlorogenic Caffeic

Extract Gentisic Acid Luteolin Apigenin
Acid Acid Acid Acid
SN1 0.09 ± 0.02 0.24 ± 0.91 2.77 ± 0.45 1.50 ± 0.66 0.64 ± 0.87 0.98 ± 0.33 0.16 ± 0.74
SN2 0.04 ± 0.05 0.19 ± 0.11 2.01 ± 0.98 1.81 ± 0.75 0.42 ± 0.54 0.8 ± 0.62 0.12 ± 0.02
Foods 2019, 8, 63
Figure 1. Representative chromatogram of the dry extract reporting the retention time (RT) of gallic
acid (1, 0.65 min), protocatechuic acid (2, 13.85 min), chlorogenic acid (3, 20.5 min), gentisic acid (4,
25.1 min), caffeic acid (5, 27.5 min), luteolin (6, 52.9 min), and apigenin (7, 70.95 min). Axis: x label
minutes, y label absorbance unit.
3.2. Antioxidant Activity in a Cellular Free System

The free radical-scavenging activity of SN1 and SN2 extracts was tested by their ability to bleach
the stable DPPH radical. Both extracts were able to quench the DPPH-radical at all the concentrations
used (0.025-0.5-0.1-0.2-0.4 mg/mL) in a dose-dependent manner. SN1 showed a more potent capacity
than SN2. In addition, their effect appeared similar to Trolox (30 μM), which is a soluble analogous of
vitamin E used as a standard. This experiment demonstrated that SN1 and SN2 possess antioxidant
properties (Figure 2).
Figure 2. Quenching of DPPH of SN1 and SN2 extracts at different concentrations (0.025-0.5-0.1-0.2-0.4
mg/mL), compared to Trolox (30 mM). Axis: x label: concentration, y label: quenching of DPPH
expressed as a percentage. (* p < 0.05 and ** p < 0.001).
3.3. Antioxidant Activity in the Cellular System

Primary rat astroglial cultures exposed to 500 μM glutamate for 24 h were used as an in vitro
cellular model to assess the antioxidant effect of SN1 and SN2 extracts. Cells were characterized by
immuno-fluorescent staining using GFAP as a marker [31].
The glutamate-evoked oxidative stress was evaluated by measuring the depletion of intracellular
GSH levels (Figure 3) and ROS production (Figure 4). Glutamate (500 μM for 24 h) produced a
significant decrease in the intracellular GSH levels and a significant increase of ROS levels, when
compared to the untreated control ones. The pre-incubation of the cultures with SN1 and SN2 extracts
Foods 2019, 8, 63
(0.5 and 1 mg/mL) was able to restore, in a dose-dependent manner, GSH and ROS levels. In particular,
1 mg/mL of the extracts showed values similar to untreated control values.
Figure 3. GSH levels in primary rat astroglial cell cultures at 14 DIV: exposed to glutamate 500 μM for
24 h. Bar 1: control. Bar 2: cell culture exposed 500 μM. Bar 3: cell culture exposed 500 μM plus SN1
0.5 mg/mL. Bar 4: cell culture exposed 500 μM plus SN1 1 mg/mL. Bar 5: cell culture exposed 500 μM
plus SN2 0.5 mg/mL. Bar 6: cell culture exposed 500 μM plus SN2 1 mg/mL. Four replicates were
carried out for each sample. (* p < 0.05 and ** p < 0.001). Axis: x label: concentration, y label: nmoli of
GSH per mg of protein.
Figure 4. ROS levels in primary rat astroglial cell cultures at 14 DIV: exposed to glutamate 500 μM
for 24 h. Bar 1: control. Bar 2: cell culture exposed 500 μM. Bar 3: cell culture exposed 500 μM plus
SN1 0.5 mg/mL. Bar 4: cell culture exposed 500 μM plus SN1 1 mg/mL. Bar 5: cell culture exposed
500 μM plus SN2 0.5 mg/mL. Bar 6: cell culture exposed 500 μM plus SN2 1 mg/mL. Four replicates
were carried out for each sample. (* p < 0.05 and ** p < 0.001). Axis: x label: concentrations, y label:
percentage of fluorescence intensity per mg protein versus control.
4. Discussion
In this study, we assessed the antioxidant effect of SN1 and SN2 extracts of Solanum nigrum L.
leaves, both in an in vitro cellular free system and in vitro cellular models.
Foods 2019, 8, 63
Oxidative stress is the causative agent in a number of human diseases, such as atherosclerosis,
ischemic reperfusion injury, inflammation, carcinogenesis, aging, and neurodegenerative diseases.
Although there are many determinants in the development of these diseases, considerable experimental
evidence links the production of ROS to biological damage that can potentially provide a mechanistic
basis for their initiation and/or progression [36–39]. Moreover, because the ROS production is the
fatal consequence of aerobic life, it is also an important component of the signaling network of
plants [39], where polyphenols are the secondary metabolites produced as a defense mechanism
against stress factors.
In this study, we exploited the sources, composition, and mechanisms of action of two polar
Solanum nigrum L. leaf extracts natural products, and food, which represent a new frontier for therapy
and a valuable tool to reduce the costs of health care systems.
In recent years, there has been great interest in the health effects of various natural products and
in the in vivo protective function of natural antioxidants contained in dietary food against oxidative
damage caused by ROS [40–45].
The free radical-scavenging activity is measured by the ability to bleach the stable DPPH radical.
This assay provided information on the reactivity of test compounds with a stable free radical. SN1
and SN2 extracts were able to quench the DPPH-radical in a dose-dependent manner and showed
comparable capacity. In fact, the two polar extracts content of different level phenolic components, but
phenolic and flavones are not significantly different between SN1 and SN2. Only gentisic acid is more
abundant in SN2. In addition, their effect appeared similar to Trolox. Then, this set of experiments
demonstrate that SN1 and SN 2 possess comparable antioxidant properties.
Furthermore, we assessed the effect of the extracts in an in vitro cellular experimental model
of excitotoxicity. In our previous research studies, using an experimental model of excitotoxicity,
we demonstrated that glutamate exposure in primary cultures of astrocytes might be part of the
biochemical response to oxidative stress induced by a prolonged exposure of astrocyte cultures to the
neurotransmitter [46].
The antioxidant effect of the extracts SN1 and SN2 was also assessed in the cellular system using
primary rat astroglial cell cultures exposed to the astroglial cell cultures in the presence of 500 μM
glutamate for 24 hours. We used glutamate as a stressor because its high levels induce alterations in
glutamate transport, mitochondria impairment, decrease ATP levels, GSH depletion, ROS production,
macromolecular synthesis [35], and subsequent neuronal cell death [47,48].
Figure 2 shows the quenching of DPPH of SN1 and SN2 extracts at different concentrations,
compared to Trolox, which shows a stronger activity at a lower concentration (0.025–0.5 and
0.1 mg/mL). In fact, we found that the extracts SN1 and SN2 were able to counteract the effect of
glutamate, restoring, in a dose-dependent manner, the GSH and ROS levels similar to the control values.
The statistical analysis method in this study indicated high significance (* p < 0.05 and ** p < 0.001)
when compared with the control group, as reported in Figures 3 and 4, where SN1 and SN2 extracts
are compared with the cells exposed to glutamate 500 μM only.
The protective effect against glutamate toxicity of the extracts SN1 and SN2 appeared stronger
than that of the synthetic antioxidant compounds used in our previous research studies [46].
Thus, these findings show that the extracts SN1 and SN2 possess antioxidant properties.
Furthermore, it is possible to assume that the extracts of SN1 and SN2 of Solanum nigrum are able to
counteract glutamate uptake-induced impairment of cystine/glutamate antiporter, which leads to
depletion of the GSH content and biochemical alterations. This results in the delayed toxic effect for
primary astroglial cell cultures [35]. Moreover, in a previous study, we reported that a pre-incubation
with GYKI 52466, the selective inhibitor of AMPA/KA receptors, diminished glutamate effects, which
indicates the involvement of receptor-linked events in GSH decrease and ROS increase levels [49].
Spectrophotometric and chromatographic analytical methods applied for estimation of total
phenolic content and for determination of phenolic acids and flavones compounds in the examined
extracts showed that these constituents are present in a valuable amount. Our results strongly suggest
Foods 2019, 8, 63
that phenolic compounds are important components of SN, and some of their pharmacological effects
could be attributed to the presence of these compounds.
5. Conclusions
This study has provided some scientific rationale for these vegetables in the management of
diseases, as obtained in folklore.
SN leave extracts were able to reduce oxidative stress, and, in particular, they showed the
potential in quenching the radical in vitro free cellular system, and restoring the oxidative status
among in vitro primary rat astroglial cell cultures exposed to glutamate, which possesses notable
antioxidant properties and neuroprotective effects. Furthermore, preventing the increase in glutamate
uptake and inhibiting glutamate excitotoxicity, SN1 and SN2 leave polar extracts may represent a new
natural therapeutic strategy in the neuro-pathological conditions associated with excitotoxicity.
Therefore, these vegetables may serve as a potential source of natural phenolic antioxidants for
functional foods and nutraceuticals for the prevention and management of neurodegenerative diseases.
Author Contributions: The CRediT taxonomy is listed below to define author contributions to primary research
papers and the independent contributions of each author is provided, with the agreement of all co-authors.
Each author on the paper had more CRediT contribution roles, as listed below: conceptualization, A.C., N.A.S.;
methodology, A.C., R.A., M.R., N.A.S.; software, G.R.; validation, N.A.S., M.R.; formal analysis, M.R., G.R.;
investigation, G.R., R.A.; resources, A.C., R.A., N.A.S., A.D.; data curation, R.A.; G.R., writing—original draft
preparation, N.A.S., A.C., R.A.; writing—review and editing, M.R.; visualization, M.R.; supervision, A.C., N.A.S.,
R.A.; project administration, A.C., R.A., N.A.S.; funding acquisition, M.R.
Conflicts of Interest: The authors declare no conflict of interest and the founder added the requested detail. The
funder M.R. had roles in methodology, validation, formal analysis, visualization and the decision to publish
the results.
Abbreviations
ATP Adenosine Triphosphate
DCF 2 ,7 -dichlorofluorescein
DCFH-DA 2 ,7 -dichlorofluorescein diacetate
DIV days in vitro
DMEM Dulbecco’s Modified Eagle’s medium
DPPH 1,1-Diphenyl-2-Picrylhydrazyl radical
EDTA ethylenediaminetetracetic acid
FBS Heat Inactivated Fetal Bovine Serum
FITC fluorescein isothiocyanate
GFAP Glial Fibrillary Acidic Protein
GSH reduced glutathione
HPLC High performance liquid chromatography
MTT 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide
NADH β-Nicotinamide adenine dinucleotide
O2 • − superoxide anion
•OH hydroxyl radical
ROO• peroxyl radical
ROS reactive oxygen species
SOD Superoxide dismutase
SN Solanum Nigrum L
XO Xanthine oxidase
Foods 2019, 8, 63
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foods
Article
Effect of a Milk-Based Fruit Beverage Enriched with
Plant Sterols and/or Galactooligosaccharides in a
Murine Chronic Colitis Model
Gabriel López-García 1 , Antonio Cilla 1, *, Reyes Barberá 1 , Amparo Alegría 1 and María C. Recio 2
1 Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia,
Avda. Vicente Andrés Estellés/n, 46100 Burjassot (Valencia), Spain; gabriel.lopez@uv.es (G.L.-G.);
reyes.barbera@uv.es (R.B.); amparo.alegria@uv.es (A.A.)
2 Department of Pharmacology, Faculty of Pharmacy, University of Valencia, Avda. Vicente Andrés Estellés/n,
46100 Burjassot (Valencia), Spain; maria.c.recio@uv.es
* Correspondence: antonio.cilla@uv.es; Tel.: +34-963-544-972
Received: 7 March 2019; Accepted: 1 April 2019; Published: 4 April 2019
Abstract: The potential anti-inflammatory effect of plant sterols (PS) enriched milk-based fruit
beverages (PS, 1 g/100 mL) (MfB) with/without galactooligosaccharides (GOS, 2 g/100 mL) (MfB-G)
in an experimental mice model of chronic ulcerative colitis was evaluated. Beverages were orally
administered to mice every day by gavage to achieve PS and GOS doses of 35 and 90 mg/kg,
respectively, and experimental colitis was induced by giving mice drinking water ad libitum containing
2% (w/v) dextran sulphate sodium (DSS) for 7 days, alternating with periods without DSS up to the
end of the study (56 days). MfB beverage showed significant reduction of symptoms associated to
ulcerative colitis and improved the colon shortening and mucosal colonic damage, but it was not able
to reduce the increase of myeloperoxidase levels produced by DSS. MfB-G showed higher incidence of
bloody feces and loss of stool consistency than MfB, as well as high levels of immune cells infiltration
in colon tissue and myeloperoxidase. Therefore, PS-enriched milk-based fruit beverage could be an
interesting healthy food to extend the remission periods of the diseases and the need to evaluate, in a
pre-clinical model, the anti-inflammatory effect of the combination of bioactive compounds in the
context of a whole food matrix.
Keywords: milk-based fruit beverage; plant sterols; galactooligosaccharides; mice; chronic

ulcerative colitis
1. Introduction
Ulcerative colitis (UC) is a chronic inflammatory disease, within intestinal bowel diseases (IBD),
characterized by a tissue destruction of large intestine with relapsing phases followed by periods of
remission [1]. The most common treatment strategies used include pharmacological therapy (steroidal
and non-steroidal drugs) or, in the worst cases, removing portions of affected gastrointestinal tract
through surgery. Although, pharmacological therapy has shown efficacies in ameliorating the severity
and symptoms of IBD, typically does not lead to long periods of remission. Consequently, recent
investigations have focused on the study of the impact of diet and healthy foods as potential alternative,
and even the possible combination of nutraceuticals and healthy foods with pharmacological therapy
in cases when the patient is not eligible for conventional therapy which could help extend remission
periods and improve life quality of IBD patients [2–6].
A recent meta-analysis has associated a high intake of fruit and vegetables with a reduction
of UC in European population, suggesting that the intake of their bioactive compounds (fiber,
antioxidant vitamins and phytochemicals such as polyphenols, carotenoids, isoflavones) could

Foods 2019, 8, 114
explain this inverse association [7]. In murine colitis models, which mimic human pathology,
it has been demonstrated the preventive effect on pro-inflammatory intestinal process associated
to the intake of mango [8] or pineapple [9] juices, polyphenols-rich extracts from orange [10] or its
sub-products [11], apple [12] and pomegranate [13]. Polyphenols seems to be the main bioactive
compounds involved in the anti-colitic action of fruits, due to its ability to inhibit some pivotal
pro-inflammatory mediators as nuclear transcriptional Factor-κB (NF-κB) and specific cytokines
(Tumor Necrosis Factor-α (TNF-α), and interleukin 1-β (IL-1β)) and the induction of antioxidant
defence systems. Furthermore, lipophilic compounds present in vegetables and fruits, such as
fat-soluble vitamins (pro-vitamin A) [14], carotenoids (β-carotene) [15] and plant sterols (PS) [16–21],
have displayed important anti-inflammatory and inmmunodulatory effects. In particular, PS could be
a good strategy to prevent IBD, because they show a very little absorption in the intestine (0.5–2%) and
reach the colon, where could exert anti-inflammatory effects. Moreover, the European Union authorized
PS addition into milk-based fruit beverages [22], among other foods, to achieve the necessary amount
of 1.5–3 g PS/day (not attainable with normal diet) for the well-known cholesterol-lowering effect [23].
PS have shown promising results in animal colitis models induced by trinitrobenzene sulfonic acid
(TNBS) [16,17], dextran sodium sulphate (DSS) [18–24] or high-fat diet (40–60%) [19–21]. Doses
between 20–150 mg/Kg/day help to mitigate some important parameters associated to UC such as
symptoms, colon shortening and presence of edema, histopathology and myeloperoxidase (MPO)
activity on colon tissue.
Other bioactive compounds that have gained attention are prebiotics, which stimulate selectively
beneficial intestinal bacteria, helping to maintain intestinal mucosal barrier and enhance defense
against pathogenic microorganism [25]. In this sense, galactooligosacharides (GOS) have been
proposed as an active ingredient to prevent or alleviate symptoms associated with IBD [26]. Human
clinical trials have demonstrated that consumption of GOS contained in chocolate or banana flavored
chews (3.5–7 g/day) [27] and sachets (5.5 g/day) [28,29], reduce different pro-inflammatory markers
such as calprotectin in feces, plasma C-reactive protein [28] and cytokines (IL-6, IL-1β and TNF-α) [29],
with a concomitant increase in levels of fecal secretory immunoglobulin A (sIgA) [28] and reduction of
common ulcerative colitis symptoms [27]. However, in animal studies the effect of GOS on colitis is
controversial. GOS (4 g/Kg/day) administration for 10 days increase bifidobacterial number in colon
in a TNBS colitis induced murine model, but it was not linked with a reduction of pro-inflammatory
markers as MPO activity and colon damage [30]. On the other hand, in mice smad-3 feed with GOS
(5 g/Kg/day) for 42 days an improvement of colitis severity and increase of natural killer activity were
observed after colitis induction by Helicobacter hepaticus [31].
Enrichment of healthy foods with bioactive compounds could be an effective strategy to help
mitigate the symptoms associated with IBD and extend remission periods [3]. In this sense, milk-based
fruit beverages could be a suitable nutritional matrix for this purpose due to its low-fat content and
good sources of bioactive compounds such as polyphenols, carotenoids and vitamins. Moreover,
previous studies of our research group have demonstrated the beneficial effect of PS enriched
milk-based fruit beverages on oxidative stress prevention and intestinal epithelia integrity maintenance
in Caco-2 cells [32], as well as, their systemic anti-inflammatory properties, in hypercholesterolemic
post-menopausal women, through the serum increase of anti-inflammatory cytokine IL-10 with
concomitant reduction of IL-1β, cytokine which play an important role on the development of IBD [33].
The present study investigates for the first time, the effect of the daily intake of a healthy food,
as milk-based fruit beverages, enriched with bioactive compounds (PS and/or GOS) on clinical
symptoms and inflammatory process of UC, using a pre-clinical chronic murine model induced by DSS.
The aim of this preliminary study is to evaluate if PS enriched milk-based fruit beverages with/without
GOS are a suitable dietetic strategy to help to mitigate the health problems associated with UC.
Foods 2019, 8, 114
2.1. PS Milk-Based Fruit Beverage Formulation

Two Ps-enriched (1 g/100 mL beverage) milk-based fruit beverages were used: MfB and MfB-G,
without or with addition of GOS (1.8 g/100 mL beverage), respectively. Both beverages contained
skimmed milk with the addition of milk fat and whey protein concentrate enriched with milk
fat globule membrane (MFGM, Lacprodan® MFGM-10 from Arla Foods Ingredients, Sønderhøj,
Denmark), mandarin juice from concentrate (45%) (Source of β-cryptoxanthin 205 μg/100 mL
beverage) and banana puree (4%). PS were added during beverage formulation as microencapsulated
free microcrystalline PS (β-sitosterol 80%; sitostanol 12%; campesterol; 7%; campestanol 1% and
stigmasterol 0.7%) from tall oil in a powder form (Lipohytol® 146 ME Dispersible from Lipofoods,
Barcelona, Spain). GOS syrup (Vivinal® GOS from Friesland Campina Ingredients, Amersfoort,
Netherland) containing approximately 59% GOS, 21% lactose, 19% glucose and 1% galactose based on
dry matter. In order to guarantee microbiological stability, beverages were subjected to pasteurization
(90 ◦ C for 30 s) and filled aseptically in 250-mL tetra bricks.
2.2. Animals and Treatment

Female C57BL/6 mice, aged 6–8 weeks with weights ranging between 18 to 20 g, provided by
Harlan Interfauna Iberica (Barcelona, Spain) were used in this study. Animals were acclimatized for
7 days under a 12-h light/ dark cycle at 22 ◦ C/60% humidity and fed with a standard laboratory rodent
diet and water ad libitum before experiments. All experiments were approved by the Institutional
Ethics Committee of the University of Valencia, Spain (2017/VSC/PEA/00143).
Chronic colitis induction was performed by repeated administration of a 2% (w/v) of DSS
(in drinking water) for 7-day cycles according to Marín et al. [34] The DSS phases were interrupted
by 7-days cycles of access to drinking water without DSS. MfB and MfB-G were orally administered
everyday by gavage (0.15 mL) to achieve 35 and 95 mg/Kg body weight for PS and GOS, respectively
(see Figure 1). Animals were randomly assigned into six intervention groups with 8 mice/group
(n = 48): control (mice received water during all experiment), DSS (access to drinking water containing
2% DSS), DSS + MfB (drinking water containing 2% DSS + 0.15 mL of MfB), DSS + MfB-G (drinking
water containing 2% DSS + 0.15 mL of MfB-G), MfB (received only 0.15 mL of MfB) and MfB-G
(received only 0.15 mL of MfB-G).
Figure 1. Experimental design of the chronic colitis induction by dextran sulphate sodium (DSS) (2%,
w/v) and plant sterol enriched milk-based fruit beverages without (MfB) or with galactooligosacharides
(MfB-G) administration in the procedures.
Water intake was monitored three times a week to guarantee equitative DSS intake among different
colitis mice groups and the type of beverages did not affect drink behavior. No statistically significant
Foods 2019, 8, 114
differences (p > 0.05) in water intake was found among different mice group (ml/mouse/day); control
(4.3 ± 0.5), DSS (4.2 ± 0.8), DSS + MfB (4.5 ± 0.7), DSS+MfB-G (4.8 ± 1.1), MfB (4.6 ± 0.4) and MfB-G
(4.5 ± 0.5).
2.3. Disease Activity Index (DAI)

The disease activity index (DAI) was determined according to the parameters outlined in Table 1.
Animal body weights, stool consistency and visible blood in feces were recorded three times a week
during all experiments. Stool consistency and blood in feces parameters were evaluated checking
fresh feces contained in each animal group cage and scored following the method described by
Marín et al. [34]. Also, blood presence around mouse perianal area was taken as indicator of blood in
feces. DAI was calculated by combining scores (WS: weight loss; SC: stool consistency; BF: bloody
feces) following the next formula DAI: (WS + SC + BF)/3 according to the methodology described by
Marín et al. [34].
Table 1. Scoring system to calculate the disease activity index (DAI *).
Score Weight Loss (%) Stool Consistency Visible Blood in Feces

0 None
Normal None
1 1–5
2 6–10 Loose Slight bleeding
3 11–20
4 <20 Diarrhea Gross bleeding
* DAI was calculated by combining scores (WS: weight loss; SC: stool consistency; BF: bloody feces).
2.4. Colon Shortening and Presence of Edema

Animals were sacrificed at day 56 by cervical dislocation and their colons (ileo-cecal junction to
anal verge) were removed. Fecal residues were washed with cold phosphate buffered saline (PBS) and
the length and weight were measured. Presence of edema in colonic tissue was evaluated working out
the weight/length (mg/cm) ratio. Then, colons were cut in four portions of approximately 2 cm and
frozen immediately at −80 ◦ C until use.
2.5. Histological Analysis

Portions of approximately 2 cm from the colon of every group of mice were fixed in freshly
prepared 4% formaldehyde in PBS buffer (pH 7.2), embedded in paraffin, and sectioned at 4 μm.
Sections were stained with hematoxylin and eosin for histopathological analysis and examined by
light microscopy [35]. The slides were analysed and scored as previously described Cooper et al. [36]
according to the criteria listed in Table 2. Scores were calculated by adding the score for the three
parameters, giving a maximum score of 10.
Table 2. Scoring system used to calculate the histological score in dextran sulphate sodium
(DSS)-induced colitis.
Histological Scoring System for DSS-Induced Colitis

Feature Score Description
0 None
1 Mild
Severity of inflammation
2 Moderate
3 Severe
0 None
1 Mucosa
Extent of inflammation
2 Mucosa and submucosa
3 Transmural
Foods 2019, 8, 114
Table 2. Cont.
Histological Scoring System for DSS-Induced Colitis

Feature Score Description
0 None
1 1/3 damages
Crypt damage
2 2/3 damaged
3 Crypts lost, surface and epithelium present
4 Crypt and surface epithelium lost
Histological score is calculated by adding the score of three parameters up to a total score of 10 points. DSS: dextran
sulphate sodium;
2.6. Myeloperoxidase (MPO) Assay

In the model of experimental colitis, neutrophil infiltration into the colon was assessed indirectly
by measuring myeloperoxidase (MPO) following the method elaborated by Bradley et al. [37]. Colon
samples were weighed and blended in homogenizer with phosphate buffer (0.22 M) containing 0.5%
of hexadecyl-trimethyl-ammonium bromide. Samples were centrifuged at 12000 g/4 ◦ C/20 min
and supernatants were placed on 96-well plates. Estimation of MPO content in colonic tissues was
evaluated spectrophotometrically (450 nm) by measuring the 3,3 ,5,5 -tetramethyl-benzidine (MPO
substrate) oxidation in presence of H2 O2 [34].

The results are expressed as means ± standard deviation values (n = 8 for each group). One-way
analysis of variance (ANOVA) followed by the Tukey post-hoc test was applied to determine differences
among treatments. A significance level of p < 0.05 was adopted for all comparisons, and the Statgraphics
Centurion XVI.I statistical package (Statpoint Technologies Inc., VA, USA) was used throughout.
3. Results
3.1. Evaluation of Clinical Symptoms of Induced Chronic Colitis in Mice

Mice exposed to DSS in drinking water (2% w/v) showed marked clinical symptoms (DAI values)
during all experiment, being slightly lower in the first DSS cycle (7 days) (see Figure 2a). Administration
of MfB significantly reduced DAI values between 24–67% compared to DSS group, with the exception
of the first DSS cycle. MfB-G beverage attenuated DSS derived symptoms during the first cycles (7 and
21 days), reducing DAI values between 40–54% with respect to the DSS group. However, from the third
DSS cycle (35 days) onwards, an increase of DAI (1.6 fold compared to the DSS group) was observed.
With the aim to evaluate the effect of beverages on specific illness derived symptoms, Figure 2b,c show
the evolution of mice weight, stool consistency and presence of blood in feces, respectively. In general,
MfB and MfB-G administration did not have any differences in terms of weight loss compared to DSS
group during the study, with the exception of MfB-G beverage, where a statistical (p < 0.05) higher
weight loss at 10 day (20% vs. DSS group) was observed. Both beverages statistically (p < 0.05) reduced
the presence of blood in feces (4 to 6-fold) during the acute phase of the illness (7 days) with respect to
the DSS group (Figure 2c). However, in subsequent DSS cycles, this protective effect was not observed
with both beverages and sometimes the incidence of blood in faces increased in the MfB-G mice group
(third and fifth cycle). With respect to stool consistency, MfB prevented completely the appearance of
diarrhea during the first two DSS cycles (7 and 21 days) and reduced up to 50% their incidence during
the third DSS cycle, compared to DSS group, (see Figure 2c). However, after fourth (49 days) DSS
cycle MfB did not show differences in the stool consistency in comparison to DSS group, although
a slight beneficial effect was observed at the end of the study. On the other hand, MfB-G partially
prevented the loss of stool consistency during the second DSS cycle, but this loss rapidly increased in
the subsequent DSS cycles after which stool consistency remained constant for the rest of the study.
The loss of stool consistency observed in the MfB-G group during the third cycle coincides with the
Foods 2019, 8, 114
increase of blood presence in feces. This suggests that the higher acute diarrhea observed is related to
colonic epithelium erosion.
DAI score (arbitrary units) 2.5 Đ Đ

2 Đ
Ă Ă
Ă Ă
1.5 ď
Ă Đ ď
1 Ăď ď
Đ ď
0.5
0
7 21 35 49 56
Days
(a)
(b)
ď
ď Ă
ď
6FRUHDUELWUDU\XQLWV
Ăď

Ă Ă

Ă Ă
Ă

Ă Ă
Ă
ď
ď

(c)
Figure 2. Effect of plant sterol enriched milk-based fruit beverages without (MfB) or with
galactooligosacharides (MfB-G) on clinical symptoms in DSS-induced chronic colitis model. (a) Disease
activity index (DAI) score after DSS (dextran sulphate sodium) administration in each cycle; (b) body
weight per mouse during all the experiments; (c) stool consistency loss (lines) and presence of blood
in feces (bars) at the end of each DSS administration cycle. Bars/markers show the mean ± standard
deviation (n = 8). Different lowercase letters (a–c) indicate statistically significant differences (p < 0.05)
among mice groups (DSS, DSS + MfB and DSS + MfB-G) within the same DSS cycle. (*) Denotes
statistically significant differences (p < 0.05), compared to the DSS group, at the same day.
Foods 2019, 8, 114
3.2. Colon Length Shortening and Presence of Edema

Figure 3a shows that mice exposed to DSS (6.10 ± 0.28 cm), DSS + MfB (7.20 ± 0.35 cm) and DSS
+ MfB-G (6.33 + 0.35 cm), suffered a significant shortening (p < 0.05) of colon length in comparison to
the control (8.53 ± 0.29 cm). DSS + MfB mice showed longer colon (18%) than DSS mice, significantly
preventing (p < 0.05) the colon shortening induced by DSS. Mice that received DSS showed higher
presence of edema in colonic tissue than the control group (39–50 vs. 28 mg/cm, respectively)
(see Figure 3b). Administration of beverages did not show any significant (p > 0.05) protective effect
with respect to edema development. Mice that received MfB and MfB-G showed very similar colon
length and tissue edema compared to control, which is according with the absence of clinical symptoms
observed on these mice groups.
10
Ă Ă
9 Ă
8 Đ
Colon length (cm)
7 ď ď
6
5
4
3
2
1
0
Control DSS DSS + MfB DSS + MfB-G MfB MfB-G
(a)
70
Colon weight/length (mg/cm)
60 Đ
ďĐ
50
ď
40 Ă
Ă Ă
30
20
10
0
Control DSS DSS + MfB DSS + MfB-G MfB MfB-G
(b)
Figure 3. Evaluation of colon length (a) and presence of edema (b) after administration of plant
sterol enriched milk-based fruit beverages without (MfB) or with galactooligosacharides (MfB-G) for
56 days on dextran sulphate sodium (DSS)- chronic colitis induction. Bars show the mean ± standard
among samples.
3.3. Histopathological Analysis

As is shown in Figure 4A, control, MfB and MfB-G mice groups (Figure 4a,c,e, respectively) had a
morphologically normal colon without signs, or very low levels of leucocyte infiltration. In contrast,
DSS group (Figure 4b) suffered a marked inflammation characterized by a loss of epithelial architecture,
reduction of number of globet cells and crypts, as well as a strong increase of immune cell infiltration
compared to control (Figure 4a), what it was reflected in a high histological score (9/10 points).
Treatment with DSS + MfB (Figure 4d) or DSS + MfB-G (Figure 4f) mice showed similar histopathology
Foods 2019, 8, 114
alteration compared to DSS group (b), with immune cell infiltration and distortion of crypts, although
the epithelial architecture was more preserved resulting in a significant (p < 0.05) lower histological
score (34 and 10%, respectively) values. However, DSS + MfB-G (Figure 4f) mice showed a higher
histological score (25%) and damage in the epithelial architecture, with distortion of crypts and high
presence of immune cell in comparison to the DSS + MfB group (Figure 4d).
(a) Control (b) DSS
(c) MfB (d) DSS + MfB
(e) MfB-G (f) DSS + MfB-G
ϭϬ Ă Đ
,ŝƐƚŽůŽŐŝĐĂůƐĐŽƌĞ
ϴ
ď
ϲ
Ϭ
^^ ^^нDĨ ^^нDĨͲ'
B
Figure 4. The representative photographs of hematoxylin and eosin staining (magnification ×200) (A)
and the corresponding score (B) of colon tissues obtained after administration of plant sterol enriched
milk-based fruit beverages without (MfB) or with galactooligosacharides (MfB-G) for 56 days on
dextran sulphate sodium (DSS)- chronic colitis induction. Data are expressed as the means ± standard
deviation of six mice in each group. Representative images of staining of colon tissues from each (n = 3)
p < 0.05 vs. normal control. Different lowercase letters (a–c) indicate statistically significant differences
(p < 0.05) among samples.
Foods 2019, 8, 114
3.4. Presence of MPO in Colonic Tissue

Similar to that observed in the histopathological analysis, mice receiving DSS showed high levels
of immune cells infiltration, raising from 2 to 3.5-fold MPO levels with respect to control (see Figure 5).
Daily administration of beverages (DSS + MfB or DSS + MfB-G groups) failed to attenuate the increase
of MPO levels induced by DSS. MfB and MfB-G mice groups had very similar MPO values with respect
to the control, suggesting that both beverages apparently do not trigger an inflammatory response in
the colon in the absence of colitis.
Ϭ͘ϯϬ
ďĐ
DWKůĞǀĞůƐ;ĂƌďŝƚƌĂƌǇƵŶŝƚƐͿ
Ϭ͘Ϯϱ
Ϭ͘ϮϬ Đ
ď
Ϭ͘ϭϱ
Ă Ă
Ϭ͘ϭϬ
Ă
Ϭ͘Ϭϱ
Ϭ͘ϬϬ
ŽŶƚƌŽů ^^ ^^нDĨ ^^нDĨͲ' DĨ DĨͲ'
Figure 5. Presence of myeloperoxidase (MPO) levels in colonic tissue after administration of plant
sterol enriched milk-based fruit beverages without (MfB) or with galactooligosacharides (MfB-G) for
56 days on dextran sulphate sodium (DSS)- chronic colitis induction. Bars show the mean ± standard
among samples.
4. Discussion
To investigate the potential anti-inflammatory effects of PS enriched milk-based fruit beverages
with or without GOS on UC, a model of chronic colitis induced by DSS in mice was selected. Although
colitis animal models do not represent the complexity of human disease, they provide valuable
information about factors involved in the inflammatory process and allow to evaluate different
therapeutic strategies to improve life quality of patients with IBD [38]. In particular, the DSS-induced
colitis model can easily reproduce the acute and chronic phases or the relapsing periods typical
of UC, depending on the concentration and cycles of exposition to DSS in drinking water. Moreover,
this model exhibits similar symptoms to those of human UC (diarrhea, bloody feces and body weight
loss) and histological features such as mononuclear leucocyte infiltration, crypt architectural disruption
and epithelial degeneration [39].
Our study showed that daily administration of MfB beverage resulted in a significant (p < 0.05)
reduction of symptoms associated to UC, mainly preventing the presence of diarrhea or alleviating
their increase during all experiment (vs. DSS group), as well as, protecting against presence of bloody
feces in the acute phase of the disease (first DSS cycle). Reduction of clinical symptoms led to partially
prevention of colon shortening induced by DSS cycles, showing longer colons (18%) compared to DSS
group. Histopathological analysis of the colon revealed a slight improvement in the architecture of
the colonic epithelium and a greater number of crypts compared to the DSS group, in agreement with
the previous parameters mentioned. However, the distribution and morphology of the crypts were
altered, as well as an increase of neutrophil infiltration and a high MPO level in the colonic tissue was
observed. Stimulation of neutrophil activity or its migration into colon tissue, could be related with the
Foods 2019, 8, 114
prevention of colon shortening observed after MfB treatment. Neutrophils are considered the first line
of defence against microorganisms and recently, it has been demonstrated that they can build a complex
formed by chromatin and neutrophil proteins that act as immunomodulator and activate immune
cells such as T cells, although their specific role on IBD has not been well described yet [40]. Beneficial
effect of MfB upon DSS-induced colitis can also be related with its cytoprotective effect at intestinal
level. In a previous in vitro study by our research group, it was demonstrated that milk-based fruit
beverages had several beneficial effects against oxidative stress and prevention of cell dead, inhibiting
some important pro-apoptotic events and preserving cell monolayer integrity in a differentiated colon
cancer (Caco-2) cell model [32]. This fact could be important because DSS is a toxic compound to
colonic epithelial cells that cause an increase of apoptotic cells and compromise the epithelial barrier
integrity through the loss of some important proteins present in the tight junctions [41].
It is important to note our study design does not allow knowing which bioactive compound/s
contained in the beverages has the anti-inflammatory effect on DSS-induced colitis. Nevertheless,
it may be possible that the specific combination of all of them produce the effect observed in our
study. The presence of mandarin juice (represents almost half of MfB composition), could contribute to
the anti-inflammatory effect since contains important quantities of antioxidant phytochemicals such
as flavonoids (mainly hesperidin, narirutin and vicenin-2) and β-cryptoxanthin (β-Cx) [42]. In this
sense, flavonoid-rich extracts (containing mainly hesperidin, narirutin and vicenin-2) obtained from
blood orange juice administrated (40 mg/kg/day) to CD1 mice with colitis-induced by dinitrobenzene
sulfonic acid, have shown preventive effects against the colonic pathological tissue damage. Flavonoids
acted mainly counteracting NF-κB signalling, decreasing expression of pro-apoptotic proteins (Bax) and
restoring the redox balance in colonic tissue [10]. Similar effects were observed in a recent study with
industrial orange by-products (citrus pectin and different sub-fractions obtained from orange after juice
extraction) in DSS-treated mice [11]. Byproducts with high polyphenol total content and antioxidant
capacity showed better anti-inflammatory effect in terms of clinical symptoms and reduction of
pro-inflammatory mediators’ expression (TNF-α, IL-1β, IL-6). On the other hand, the presence of β-Cx
could contribute to the beneficial effects of the MfB, although its specific role on UC has not been
studied yet. In steatohepatitis and insulin resistance murine models induced by high fat content diets,
β-Cx administration (~2.5–7.5 mg/kg/day) lead to attenuation of lipotoxicity-induced inflammation,
preventing hepatic tissue peroxidation (TBARS) and the macrophages activation [43], as well as the
stimulation of antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) and
inhibition of the expression of pro-inflammatory markers (NF-κB and TNFα) in liver [44]. Therefore,
β-Cx is able to reduce the pro-inflammatory process through a direct and indirect anti-oxidant
mechanism. Taking into account that oxidative stress has a pivotal role on UC, daily administration of
MfB containing β-Cx (0.02 mg/kg/day) could help to mitigate the pro-inflammatory process.
Additionally, it is important to note that enrichment of MfB with PS could be a remarkable factor
in its anti-colitic effect, since several studies using PS standard solutions (β-sitosterol, stigmasterol and
γ-oryzanol) added to feed and administered at doses between 20–50 mg/kg/day (similar to our study
35 mg/kg/day) for 3-56 days have shown a marked anti-inflammatory effect independently of the
colitis animal model used. In C57BL/6 mice, β-sitosterol (20 mg/kg/day) administration for 56 days
prevented the colon shortening (~8%) and reduced MPO activity in colon tissue (~35%), what led to a
lower level of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) after colitis induction by high fat
diet (60 Kcal% from fat) [16]. Similarly, β-sitosterol administration (10 or 20 mg/kg) during 3 days
to C57BL/6 mice with colon inflammation induced by TNBS prevented partially colon shortening
(~3.4%) and improved the pro-inflammatory status, reducing pro-inflammatory cytokine levels (IL-1β,
TNF-α and IL-6) and MPO activity in colon tissue (~42%), with a concomitant inhibition of NF-κB
translocation into the nucleus [17]. In UC models induced by DSS, administration to C57BL/6 mice of
γ-oryzanol (50 mg/Kg/day for 16 days), a mixture of phytosteryl ferulates derived from rice bran oil,
mitigated clinical symptoms associated to UC and partially prevented colon shortening (~9%) and
the pathophysiological activity during colonic inflammation through inhibition of NF-κB activation
Foods 2019, 8, 114
after 5 days of DSS at 3% (v/v) induction [24]. Differences observed in terms of MPO activity with
respect to our study, could be attributed to the fact that PS are added into the beverages as a food
ingredient composed of a complex mixture of PS (β-sitosterol, sitostanol, campesterol, campestanol
and stigmasterol). As far as we know, there are no studies that evaluate the anti-inflammatory
effect—in murine chronic colitis models—of some of the PS found in the food ingredient used
in the PS enrichment of our beverages (sitostanol, campesterol and campestanol), which suppose
20% of the total PS. The small molecular structural differences among sitostanol, campesterol or
campestanol, in comparison to β-sitosterol, could explain the lack of beneficial effect observed on the
pro-inflammatory process. Feng et al. [21] observed that β-sitosterol or stigmasterol administration
(0.4% w/w) in C57BL/6J mice similarly mitigate inflammation severity and macroscopic damage (colon
shortening and histopathology score) induced by DSS (1.5% w/v, for 5 days), but only stigmasterol was
able to reduce the expression of cyclooxygenase-2. Authors indicate that the presence of a double-bond
in the side chain in stigmasterol could be responsible for their additional anti-inflammatory effect
compared to β-sitosterol. In this sense, different behaviour of β-sitosterol, campesterol and stigmasterol
at the same concentration (24 μM for 48 h) and cell system (macrophages) have been observed.
Stigmasterol suppressed cytokine secretion into the supernatant, while β-sitosterol promoted it and
campesterol did not have any effect [45]. Moreover, it is also possible that some specific compounds
present in our beverages hidden the beneficial effects of PS on colitis. Llewellyn et al. [46] observed that
administration of a high casein diet (41% w/w) for 14 days promoted intestinal barrier damage and
increased colonic cytokines levels (IL-6 and TNF-α) in DSS induced model in mice (3% w/v for 7 days).
Although, the casein intake is around 850-fold higher than our study, the longer administration time
of beverage (14 vs. 56 days) and DSS (7 vs. 56 days) exposition could explain our results. Besides,
the potential impact of other compounds present in the beverage on the colitis process cannot be
ruled out.
Regarding the potential beneficial effect of GOS on DSS-induced colitis, results show that MfB-G
administration during all the experiment does not confer additional beneficial effects with respect
to MfB. DSS + MfB-G mice suffered a dramatic increase of clinical symptoms from the third DSS cycle,
remaining constant up to the end of the study. This fact could explain the absence of a beneficial
effect of MfB-G on colon shortening (12% shorter than MfB), the higher colonic mucosa alteration
(distortion of crypts and high immune cells infiltration) and MPO level compared to MfB. The effect of
GOS and mechanisms which could improve UC in murine colitis models have been poorly studied
and currently are controversial. Holma et al. [30] reported that administration to rats of two kinds
of GOS (whey and lactose derived from cow milk) at 4 g/kg/day for 10 days increased notably
bifidobacterial number in feces, which are high producers of anti-inflammatory compounds, such as
short-chain fatty acids. However, the increase of bifidobacterial number was not correlated with an
improvement of colonic damage and they did not prevent immune cell infiltration (MPO activity) and
edema induced by TNBS during 72 h. In contrast, in mice deficient in smad-3 (phenotype characterized
by colon moderate inflammatory response) infected by Helicobater hepaticus, GOS supplementation
(5 g/kg/day) for 42 days reduced colitis severity preserving colon architecture by modulating the
function and trafficking of natural killer cells [31]. The use of different animal species, agents of
induction of colitis, treatment times and type of GOS could explain the differences found between
the studies. However, it has been reported previously the harmful effect of fructooligosacharides
(FOS) administration (at 6 g/day), a kind of soluble fibre similar to GOS, in rats with colitis induced
by DSS (at 3% for seven days). Similar to our results, FOS slightly prevented symptoms associated
to UC (13–45%, DAI value) at the beginning of the study but quickly worsened without showing
differences with respect to DSS group up to the end of the study. FOS treatment did not prevent the
colon shortening and exacerbated colon histological damage severity and MPO activity, as well as
reduced crypt cell proliferation in the distal colon, which is an integral part of the colon repair process.
Authors indicated that FOS delayed the onset of repair, promoting the harmful effect of DSS on colon
epithelia [47]. Other possible hypothesis could be related with the bifidogenic effect of FOS, what lead
Foods 2019, 8, 114
to the increase of bifidobacterial genus in the colon. It has been indicated that a quick rate of FOS
fermentation in the cecum produce an overproduction of organic acids as lactic and acetic acids [48],
which can damage the intestinal epithelium, leading to an increase of colonic permeability [49]. Similar
to FOS, it has been indicated that GOS fermentation (72 h) lead to a quick increase and accumulation
of lactic and acetic acids in the colon, compared to propionic and butyric acids, in a dynamic in vitro
colon model (TIM-2). These increases were correlated with an increase of bifidobacteria and lactobacilli
genus [50]. Then, we hypothesized that MfB-G administration could produce a change in intestinal
microbiota by increasing the organic acids that produce bacteria, which could exacerbate the loss
of the epithelial barrier integrity and the colonic mucosal permeability induced by DSS. Moreover,
the potential inhibitory effect of GOS on the colonic repair mechanism cannot be ruled out.
In summary, PS-enriched milk-based fruit beverage (MfB) shows a moderate anti-inflammatory
effect, helping to alleviate the clinical symptoms associated to colitis, the colon shortening and colonic
damage in a DSS-induced mice model of chronic colitis. Neutrophil infiltration in colonic tissue
and MPO level remained higher after MfB treatment, suggesting that they could be involved in its
anti-inflammatory action as a compensatory mechanism trying to overcome the damage induced
by DSS. GOS addition to the MfB did not show any additional beneficial effects in comparison with
MfB and even exacerbated the pro-inflammatory action induced by DSS. The higher DAI value, colonic
mucosa damage, immune cell infiltration and MPO level, suggest that presence of GOS in colon
compromise colonic epithelial permeability or delay the reparation colonic mechanism, promoting the
cytotoxic effect of DSS on colon cells.
Our results demonstrate the importance to evaluate the biological effects of bioactive compounds
in the context of a complex food matrix. PS-enriched foods could be a suitable strategy to extend
remission periods and improve the quality of life of patients with UC, but further investigation is
needed to confirm the beneficial role of PS in a food matrix on the UC.
Author Contributions: Conceptualization and funding acquisition: A.C., R.B., and A.A.; methodology: M.C.R.;
analysis and experiments: G.L.-G., and M.C.R.; writing, review and editing: G. L-G., A.C., R.B., A.A., and M.C.R.
Funding: This research was funded by Spanish Ministry of Economy and Competitiveness, through National
Project AGL2015-68006-C2-1-R (MINECO-FEDER). G.L.-G. holds a grant (ACIF/2016/449) from the Generalitat
Valenciana (Spain).
Acknowledgments: The authors want to express their acknowledgement to the Central Service for Experimental
Research (SCSIE) of the University of Valencia, in particular to Animal Production and Microscopy sections,
for their support.
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foods
Article
Anti-Obesity Effect of Extract from Nelumbo Nucifera
L., Morus Alba L., and Raphanus Sativus Mixture in
3T3-L1 Adipocytes and C57BL/6J Obese Mice
Wan-Sup Sim 1 , Sun-Il Choi 1 , Bong-Yeon Cho 1 , Seung-Hyun Choi 1 , Xionggao Han 1 ,
Hyun-Duk Cho 2 , Seung-Hyung Kim 3 , Boo-Yong Lee 4 , Il-Jun Kang 5 , Ju-Hyun Cho 2, * and
Ok-Hwan Lee 1, *
1 Department of food science and biotechnology, Kangwon National University, Chuncheon 24341, Korea;
simws9197@naver.com (W.-S.S.); docgotack89@hanmail.net (S.-I.C.); bongyeon.cho92@gmail.com (B.-Y.C.);
zzaoszz@naver.com (S.-H.C.); xionggao414@hotmail.com (X.H.)
2 Haram Co. Ltd. Jeungpyeong 27914, Korea; hansol305@naver.com
3 Institute of Traditional Medicine and Bioscience, Daejeon University, Daejeon 34520, Korea; sksh518@dju.kr
4 Department of Food Science and Biotechnology, CHA University, Seongam, Gyeonggi 13488, Korea;
bylee@cha.ac.kr
5 Department of Food Science and Nutrition, Hallym University, Chuncheon 24252, Korea;
ijkang@hallym.ac.kr
* Correspondences: dusvnd608@hanmail.net (J.-H.C.); loh99@kangwon.ac.kr (O.-H.L.);
Tel: +82-43-217-1077 (J.-H.C.); +82-33-250-6454 (O.-H.L.); Fax: +82-43-217-1088 (J.-H.C.);
+82-33-259-5561 (O.-H.L.)
Received: 3 May 2019; Accepted: 17 May 2019; Published: 19 May 2019
Abstract: The antioxidant and anti-adipogenic activities of a mixture of Nelumbo nucifera L.,
Morus alba L., and Raphanus sativus were investigated and their anti-obesity activities were established
in vitro and in vivo. Among the 26 different mixtures of extraction solvent and mixture ratios, ethanol
extract mixture no. 1 (EM01) showed the highest antioxidant (α,α-Diphenyl-β-picrylhydrazyl,
total phenolic contents) and anti-adipogenic (Oil-Red O staining) activities. EM01 inhibited lipid
accumulation in 3T3-L1 adipocytes compared to quercetin-3-O-glucuronide. Furthermore, body, liver,
and adipose tissue weights decreased in the high-fat diet (HFD)-EM01 group compared to in the
high-fat diet control group (HFD-CTL). EM01 lowered blood glucose levels elevated by the HFD.
Lipid profiles were improved following EM01 treatment. Serum adiponectin significantly increased,
while leptin, insulin growth factor-1, non-esterified fatty acid, and glucose significantly decreased
in the HFD-EM01 group. Adipogenesis and lipogenesis-related genes were suppressed, while fat
oxidation-related genes increased following EM01 administration. Thus, EM01 may be a natural
anti-obesity agent.
Keywords: anti-obesity; Nelumbo nucifera L., Morus alba L., Raphanus sativus; 3T3-L1 adipocyte;
C57BL/6J mice
1. Introduction
Obesity is defined as excessive weight gain, particularly inordinate fat accumulation in the
body [1]. Due to rapid economic growth and westernized diets, the number of patients with obesity
has increased [2], resulting in increased rates of diseases such as type 2 diabetes [3], hypertension [4],
hypercholesterolemia [5], and hyperlipidemia [6].
Obesity occurs because of a combination of overeating, lack of exercise, and neurotransmitters,
drugs, and genetic factors [7]. To improve obesity, pharmacological treatment has been applied.

Foods 2019, 8, 170
However, because these treatments involve diverse side effects such as diarrhea and vomiting,
the development of anti-obesity materials from natural products with few side effects is required [8,9].
Nelumbo nucifera L contains several flavonoids and alkaloids and has recently been used as a plain
or blended tea to treat obesity in China [10]. Morus alba L is abundant in polyphenols such as rutin and
quercetin and has been used to treat dyslipidemia, diabetes, fatty liver disease, and hypertension [11].
Raphanus sativus is an annual herb belonging to the cruciferous family and has been reported to be
effective for treating hyperlipidemia by decreasing blood sugar level [12]. Some researchers evaluated
the anti-obesity effects of Eriobotrya japonica and N. nucifera in adipocytes and obese mice using a single
material or mixture, but few studies have examined mixtures of three natural materials [13]. Because
each material has a different physiological activity, it is necessary to confirm the physiological activities
of each according to the type and mixing ratio. These materials may exert synergistic effects, but there
might be also negative effects [14,15].
In this study, we blended N. nucifera L., M. alba L., and R. sativus in different mixing ratios
and selected the optimal mixed material based on antioxidant and anti-adipogenic experiments.
The anti-obesity effects of optimal mixed material were evaluated in 3T3-L1 adipocytes and C57BL/6J
obese mice.
2.1. Sample Preparation

Nelumbo nucifera leaves, M. alba leaves, and Dried R. sativus root were supplied by Haram Co.
Ltd. (Jeungpyeong, Korea) and freeze-dried and mixed in 13 ratios. The mixing ratio of N. nucifera L,
M. alba L, R. sativus is 80:20:0 (M1), 70:30:0 (M2), 60:40:0 (M3), 50:50:0 (M4), 80:0:20 (M5), 70:0:30 (M6),
70:20:10 (M7), 60:30:10 (M8), 60:20:20 (M9), 50:30:20 (M10), 100:0:0 (M11), 0:100:0 (M12), 0:0:100 (M13).
These materials were extracted by boiling and reflux with hot water (WM) and 70% ethanol (EM)
at 60 ◦ C for 12 h. Then they were filtered, concentrated by a vacuum evaporator (Rotavapor R-200;
Buchi, Flawil, Switzerland), and freeze-dried (Bondiro; Il Shin Lab Co. Ltd., Seoul, Korea) to obtain a
powder [16].
2.2. Antioxidant Activity Analysis

We performed a DPPH (α,α-Diphenyl-β-picrylhydrazyl) assay as described previously with some
modifications [17]. First, 0.8 mL of 0.4 mM DPPH solution was added to 0.2 mL of the sample, and the
mixture was incubated in the dark for 10 min. Next, it was measured at the 517 nm absorbance using a
microplate reader (Molecular Devices, Sunnyvale, CA, USA).
DPPH radical scavenging activity (%) = (1−(Aexperiment /Acontrol )) × 100 (1)
Aexperiment : Absorbance of experimental group, Acontrol : Absorbance of control group.

The total phenolic contents were determined using Folin-Ciocalteu’s colorimetric method [18].
First, 1 mL of sample was added to 10% Folin-Ciocalteu reagent and 2% Na2 CO3 reagent in order and
incubated for 1 h in the dark. It was measured at the 750 nm absorbance using a microplate reader.
Gallic acid was used as a standard and the total phenolic contents were calculated from the standard
calibration curve (y = 19.12x − 0.0261, R2 = 0.9992).
2.3. Cell Culture and Differentiation

3T3-L1 preadipocytes were obtained from American Type Culture Collection (CL-173, ATCC,
Manassas, VA, USA) and grown in culture plates containing Dulbecco’s modified Eagle’s
medium (Lonza, Basel, Switzerland) supplemented with 10% bovine serum (Gibco, Grand Island,
NY, USA) and 1% penicillin/streptomycin (P/S; Gibco) kept at 37 ◦ C and 5% CO2 incubator
condition. 3T3-L1 preadipocytes were differentiated to adipocytes at the 2 day after confluence
by exchanging with MDI medium (DMEM containing 10% fetal bovine serum (Gibco), 1% P/S,
Foods 2019, 8, 170
0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, Saint Louis, MO, USA), 1 μM dexamethasone

(Sigma-Aldrich), and 1 μg/mL insulin (Gibco)). Every 2 days during incubation, the culture medium
was changed to DMEM containing 10% fetal bovine serum and 1 μg/mL insulin with extracts until
day 6 [19].
2.4. Cell Viability Assay

3T3-L1 preadipocytes were plated into 96-well plates (1 × 106 cells/well) and differentiated with
MDI medium along with 100 μg/mL extracts since the 2 day after confluence for 6 days. At the
day 6, Mixed XTT (2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide) and
PMS (N-methyl dibenzopyrazine methyl sulfate reagents) (WelGene, Seoul, Korea) were included into
the medium and incubated for 4 h at 37 ◦ C. Then the soluble formazan salt generated in the medium
was measured at 450 nm against 690 nm using a microplate reader [20].
2.5. Oil-Red O Staining Assay

The amount of lipid accumulation of 3T3-L1 cells differentiated on a 24-well plate for 6 days
was determined using Oil-Red O staining. In brief, the cells were washed with phosphate-buffered
saline (PBS; Gibco) and fixed in 10% formaldehyde in distilled water for 1 h. Next, the cells were dried
with 60% isopropanol and stained with Oil-Red O (Sigma-Aldrich) solution for 1 h, and then washed
with distilled water. The stained lipids were eluted with 100% isopropanol and measured using a
microplate reader at 490 nm.
2.6. Animal Experiment Design

Eight-week-old male C57BL/6J mice were obtained from Korea BioLink (Eumseong, Korea) and
maintained under standard light (12:12-h light/dark cycle), temperature (22 ± 2 ◦ C) and humidity (55 ±
15%) conditions. The diets included a normal diet (AIN-76A; Research Diets, Inc., New Brunswick,
NJ, USA) and high-fat diet (HFD; D12492; Research Diets, Inc.), and obesity was induced in the mice
fed an HFD for 2 weeks. After 2 weeks, the mice were randomly divided into eight groups; C57BL/6J
normal group, 60% kcal HFD control group, positive control group (Garcinia cambogia, GC_245 mg/kg),
EM11_100 mg/kg group, EM12_100 mg/kg group, EM01_50 mg/kg group, EM01_100 mg/kg group,
quercetin-3-O-glucuronide (Q3OG) 10 mg/kg group; n = 10). Meanwhile, Garcinia cambogia is an
ingredient in dietary supplements for weight loss [19]. These test substances were taken orally with
saline solution once a day for 8 weeks. The mice had free access to food and water, and their body
weight and calorie intake were measured weekly. Biochemical measurements were analyzed after
treatment of the candidate materials for 8 weeks and glucose tolerance test were performed 3 days later.
Experiments were carried out with the approval of the Institutional Animal Care and Use Committee
of Kangwon University for the ethical and scientific feasibility study and effective management of
animal experiments (Permit No. KIACUC-12-0140).
2.7. Glucose Tolerance Test

After administering the anti-obesity candidate materials for 8 weeks, mice were fasted for 12 h and
the blood glucose level was measured at 0, 15, 30, 45, 60, and 75 min after glucose (1 g/kg) peritoneal
administration. Blood for glucose measurement was obtained from the tail vein of mice and measured
using a serum analyzer (Accutrend plus GCTL Cobas Roche, Basel, Switzerland).
2.8. Serum Biochemical Parameter Analysis

After administering the anti-obesity candidate materials for 8 weeks, blood samples were collected
by cardiac puncture using centrifugation (2000× g, 4 ◦ C, 15 min) and stored at −74 ◦ C. Alanine
aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine, which are indices of liver
and kidney function, and total cholesterol (TC), high-density lipoprotein cholesterol (HDL-cholesterol),
Foods 2019, 8, 170
low-density lipoprotein cholesterol (LDL-cholesterol), triglyceride (TG), non-esterified fatty acid

(NEFA), and glucose, which are indices of lipid content, were measured using a biochemical automatic
analyzer (Hitachi-720, Hitachi Medical, Tokyo, Japan).
2.9. Serum Adipokine Analysis

Also, adiponectin, leptin, and insulin-like growth factor-1 (IGF-1) were isolated in blood samples
collected by cardiac puncture after administration of anti-obesity candidate materials for 8 weeks.
Each antibody was diluted in coating buffer and coated on a microwell for overnight incubation at
4 ◦ C. Each well was washed three times with washing buffer, and 100 μL of serum was dispensed,
incubated at room temperature for 1 h, and washed twice with washing buffer. Thereafter, 100 μL of
the antibody avidin-horseradish peroxidase conjugate was added and incubated at room temperature
for 1 h, and then washed again. The TMB substrate was dispensed in 100 μL, incubated in the dark for
30 min, and treated with 50 μL of stop solution, after which absorbance was measured at 450 nm.
2.10. Real-Time Polymerase Chain Reaction (RT-PCR)

Total cellular RNA was extracted from the liver, epididymal, and abdominal subcutaneous
adipose tissue using a homogenizer and Trizol reagent (Sigma-Aldrich). Total RNA was used
for cDNA synthesis with the One-step SYBR Green PCR kit (AB Science, Avenue George V,
France). The Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City,
CA, USA) used for real-time quantitative PCR. The probes containing the fluorescence reporter
dye 6-carboxy-fluorescein (Applied Biosystems) was used to indicate mRNA gene expression.
The mouse glyceraldehyde-3-phosphate dehydrogenase probe (VIC/MGB probe, primer limited,
4352339E, Applied Biosystems) was used as an internal standard. The sequence of the used
primer was as follows; Leptin sense (5 -AACCCTTACTGAACTCAGATTGTTAG-3 ) and antisense
(5 -TAAGTCAGTTTAAATGCTTAGGG-3 ); PPARγ sense (5 -ATGCCATTCTGGCCCACCAACTT-3 )
and antisense (5 -CCCTTGCATCCTTCACAAGCATG-3 ); PPARα sense
(5 -GCCTGTCTGTCGGATGT-3 )
and antisense (5 -GGCTTCGTGGATTCTCTTG-3 );
Adiponectin sense (5 -TTCAAATGAGATTGTGGGAAAAT-3 ) and
antisense (5 -ACCGATACAGTACAGTACAGTA-3 ); UCP1 sense
(5 -CGACTCAGTCCAAGAGTACTTCTCTTC-3 ) and antisense (5 -GCCGGCTGAGATCTTGTTTC-3 );
UCP2 sense (5 -TTCAAATGAGATTGTGGGAAAAT-3 ) and antisense
(5 -ACCGATACAGTACAGTACAGTA-3 ); ACX1 sense (5 -CAGGAAGAGCAAGGAAGTGG-3 )
and antisense (5 -CCTTTCTGGCTGATCCCATA-3 ); DGAT1 sense
(5 -TGCTACGACGAGTTCTTGAG-3 ) and antisense (5 -CTCTGCCACAGCATTGAGAC-3 ); SCD1

sense (5 -CATCGCCTGCTCTACCCTTT-3 ) and antisense (5 -GAACTGCGCTTGGAAACCTG-3 );

SREBP1c/ADD1 sense (5 -AGCCTGGCCATCTGTGAGAA-3 ) and antisense
(5 -CAGACTGGTACGGGCCACAA-3 ); ACS1 sense (5 -TCCTACAAAGAGGTGGCAGAACT-3 )

and antisense (5 -GGCTTGAACCCCTTCTGGAT-3 ); CPT1b sense

(5 -GTCGCTTCTTCAAGGTCTGG-3 ) and antisense (5 -AAGAAAGCAGCACGTTCGAT-3 ); FAS
sense (5 -CTGAGATCCCAGCACTTCTTGA-3 ) and antisense (5 -GCCTCCGAAGCCAAATGAG-3 );
GAPDH VIC (5 -TGCATCCTGCACCACCAACTGCTTAG-3 ). The PCR conditions were 50 ◦ C for
2 min, 94 ◦ C for 10 min, 95 ◦ C for 15 s, and 45 ◦ C for 1 min of 40 cycles.

The results are expressed as the mean ± standard deviation of triplicate experiments. An analysis of
variance and Duncan’s multiple range tests were used to determine the significance at the p < 0.05 level.
Foods 2019, 8, 170
3. Results
3.1. Effects of 26 Extracts by Mixture Ratio of N. Nucifera L., M. alba L., R. Sativus on the Antioxidant and
Anti-Adipogenic Activities
α,α-Diphenyl-β-picrylhydrazyl (DPPH) is a very stable free radical and representative reaction
substance used to measure antioxidant capacity. DPPH radical scavenging activity assay is a method
that utilizes the principle that a purple compound is discolored as yellow when radicals are eliminated
through hydrogen donation in a phenolic compound or flavonoid with a hydroxyl radical (-OH) [21].
For the extraction method, the radical scavenging activities of ethanol extracts were superior to those
of hot water extracts. For the mixing ratio, EM05 (84.30%), EM06 (83.00%), and EM01 (81.65%) were
superior in order, but there was no significant difference between these samples (Figure 1A).
(A)
(B)
Figure 1. Cont.
Foods 2019, 8, 170
(C)
(D)
Figure 1. Effects of 26 extracts by mixture ratio of Nelumbo nucifera L., Morus alba L., and Raphanus sativus
on antioxidant activity, cell viability, lipid accumulation. (A) DPPH radical scavenging activity. (B) Total
phenolic contents. (C) Post-confluent 3T3-L1 preadipocytes were differentiated along with the treatment of
each extracts for 6 days. XTT (2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide)
and PMS (N-methyl dibenzopyrazine methyl sulfate reagents) mixture was added to the medium. After
4 h of incubation, cell viability was found out by calculating the absorbance at 450 nm against 690 nm.
(D) Stained lipids were extracted and quantified by calculating the absorbance at 490 nm. Data are
presented as the mean ± SEM (n = 3). Means with different letters on bars indicate that there is a significant
difference at p < 0.05 by Duncan’s multiple range test.
The content of phenol, a representative antioxidant, was highest in EM03 (49.00 mg GAE/g), EM02
(48.40 mg GAE/g), and EM01 (47.90 mg GAE/g), with no significant difference between these samples
(Figure 1B).
Foods 2019, 8, 170
No cytotoxicity or changes in morphology were observed at the concentration of 100 μg/mL

mixtures by XTT assay (Figure 1C). Next, the anti-adipogenic activity was measured by Oil-Red O
staining, and EM01 (75.30%) was found to be the most effective mixture for inhibiting lipid accumulation
among the 26 mixtures (Figure 1D). Therefore, we selected EM01 as an optimal mixture and carried
out anti-obesity experiments using in vitro and in vivo models.
3.2. Effect of EM01 on Lipid Accumulation

We previously found that quercetin-3-O-glucuronide is a bioactive compound in mixed
materials [16]. In antioxidant and anti-obesity experiments, EM01 (100 μg/mL) was measured
to determine its anti-obesity activity with quercetin-3-O-glucuronide (7.8 μM). As shown in Figure 2,
EM01 (80.73%) showed better lipid accumulation inhibitory activity than the single materials,
EM11 (84.92%), EM12 (86.39%), and single bioactive compound treatment (84.65%). There may
have been a synergistic interaction in EM01 between quercetin-3-O-glucuronide and other compounds.
Figure 2. Effect of EM01 and its bioactive compound on lipid accumulation. Post-confluent 3T3-L1
preadipocytes were differentiated along with the treatment of each extracts and its bioactive compound
for 6 days. Stained lipids were eluted and quantified by calculating the absorbance at 490 nm. Data
are presented as the mean ± SEM (n = 3). Means with different letters on bars indicate that there is a
significant difference at p < 0.05 by Duncan’s multiple range test.
3.3. Effects of EM01 on Body Weight, Food Intake, FER, Organ Weight, and Adipose Tissue Weight in
HFD-Induced Obese Mice
There was no significant difference in the initial body weight between experimental groups,
but the final body weights in different groups were significantly different. The body weight increased
significantly in the HFD-CTL group compared to in the ND group, suggesting that obesity was induced
by the HFD. The HFD-EM01 group (100 μg/mL) showed a higher weight loss rate than the HFD-CTL
group (Figure 3A). Food intake was not significantly different between groups except for the ND group,
and the food efficiency ratio (FER) was significantly decreased in the HFD-EM01 group (100 μg/mL)
(Figure 3B,C).
Foods 2019, 8, 170
(A)G (B)G
(C)G (D)G
(E)G
Figure 3. Effects of EM01 on (A) body weight, (B) food intake, (C) FER, (D) organ weight, and (E)
adipose tissue fat weight in high-fat diet (HFD)-induced obese mice. GC, Garcinia cambogia extract;
HFD, mice were fed a high-fat diet (60% kcal fat); ND, mice were fed a normal diet (10% kcal fat); FER,
food efficiency ratio (total weight gain/total food intake × 100). Data are presented as the mean ± SEM
(n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. HFD-CTL.
The kidney and spleen weights were not significantly different among groups (Figure 3D). Liver
weight increased significantly in the HFD-CTL group compared to in the ND group, and weight
decreased significantly in the HFD-EM01 and HFD-Q3OG groups compared to in the HFD-CTL
group. According to Figure 3E, the weight of the kidney adipose tissue significantly increased in
the HFD-CTL group compared to in the ND group, but no significant inhibitory effect was observed
in any group. The weight of abdominal subcutaneous fat, epididymal, and intestine adipose tissue
Foods 2019, 8, 170
increased significantly in the HFD-CTL group compared to in the ND group, but there was an inhibitory
effect on fat accumulation in the HFD-EM01 and HFD-Q3OG group compared to the positive control
(HFD-GC group).
3.4. Effects of EM01 on glucose tolerance in HFD-induced obese mice

When glucose tolerance occurs, blood glucose levels do not rise despite glucose administration,
which is common in obese patients [22]. To investigate the effect of EM01 administration on
glucose-induced hyperglycemia, glucose was orally administered, and the glucose tolerance test
was performed over time. Blood glucose levels in all groups increased after 30 min of glucose injection.
After 60 min of glucose injection, blood glucose levels in EM01 and Q3OG administrated groups
dropped markedly even close to ND-group. On the other hand, it was confirmed that blood glucose
levels did not decrease rapidly in EM11 and EM12 administrated groups (single material) (Figure 4).
Figure 4. Effects of EM01 on glucose tolerance in HFD-induced obese mice. Data are presented as the
mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001
vs. HFD-CTL.
3.5. Effects of EM01 on the Serum Lipid Profile in HFD-Induced Obese Mice
The HFD-CTL group showed significant increases in all parameters of the serum lipid profile
compared to the ND group. The TC level was significantly decreased in the HFD-EM01 and HFD-Q3OG
groups compared to in the HFD-CTL group (Figure 5A). HDL-cholesterol was lowered by more than
LDL-cholesterol in these groups (Figure 5B,C). TC may decrease when LDL-cholesterol levels, often
referred to as bad hormones, are suppressed [23]. The TG level was higher in the HFD-CTL group
than in the ND group, but there was no significant difference between all groups compared to
HFD-CTL (Figure 5D). AST, ALT, and creatinine are used as liver toxicity marker [24] and their levels
were significantly lowered by EM01 administration (Figure 5E,F). This suggests that the 8-week oral
administration did not affect liver and kidney toxicity in obese mice.
Foods 2019, 8, 170
(A) (B)
(C) (D)
(E) (F)
Figure 5. Effects of EM01 on serum lipid profile in HFD-induced obese mice. (A) Total cholesterol.
(B) HDL cholesterol. (C) LDL cholesterol. (D) Triglyceride. (E) Creatinine. (F) AST & ALT. Data are
presented as the mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01
and *** p < 0.001 vs. HFD-CTL. AST, aspartate aminotransferase; ALT, alanine aminotransferase.
3.6. Effects of EM01 on the Energy Balancing Metabolism in HFD-Induced Obese Mice
Adipokine is a hormone specifically secreted from adipose tissue that affects endocrine system
function. We analyzed adiponectin, leptin, IGF-1, which plays an important role in normal growth and
Foods 2019, 8, 170
health maintenance, NEFA, and glucose, which is associated with energy homeostasis [25]. Adiponectin
levels in the serum were significantly decreased in the HFD-CTL group compared to in the ND group,
but there was a significant increase in the HFD-EM01 and HFD-Q3OG groups compared to in the
HFD-CTL group (Figure 6A). Leptin, IGF-1, NEFA, and glucose levels in the serum were significantly
increased in the HFD-CTL group compared to in the ND group, while HFD-EM01 and HFD-Q3OG
group were significantly decreased compared to the HFD-CTL group (Figure 6B–E).
(A) (B)
(C) (D)
(E)
Figure 6. Effects of EM01 on the energy balancing metabolism in HFD-induced obese mice.
(A) Adiponectin. (B) Leptin. (C) IGF-1. (D) NEFA. (E) Glucose. Data are presented as the mean ±
SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs.
HFD-CTL. IGF-1, insulin-like growth factor-1; NEFA, non-esterified fatty acid.
Foods 2019, 8, 170
3.7. Effects of EM01 mRNA Expression Level of Lipid Metabolism-Related Genes in HFD-Induced Obese Mice
We analyzed the mRNA levels of adipogenesis, lipogenesis, and fatty acid oxidation-related
genes in the liver, epididymal adipose tissue, and abdominal subcutaneous fat after 8 weeks of EM01
administration (Figure 7).
As shown in Figure 7A, the mRNA levels of FAS, DGAT1, SCD-1, leptin, SREBP1c, PPARγ in
the liver were significantly increased in the HFD-CTL group but decreased in the HFD-EM01 and
HFD-Q3OG groups. The mRNA levels of COX1, adiponectin, UCP2, and PPARα increased in the
HFD-EM01 and HFD-Q3OG groups compared to in the HFD-CTL group.
As shown in Figure 7B, the mRNA levels of the FAS, leptin in the epididymal adipose tissue
increased in the HFD-CTL group, but markedly decreased in HFD-EM01 and HFD-Q3OG groups.
The mRNA levels of the ACS1, ACOX1, CPT1b, UCP2, adiponectin, PPARα increased in HFD-EM01
and HFD-Q3OG groups compared to those in the HFD-CTL group.
As shown in Figure 7C, the mRNA level of UCP1 in abdominal subcutaneous fat significantly
increased in the HFD-EM01 and HFD-Q3OG groups compared to in the HFD-CTL group.
(A)
Figure 7. Cont.
Foods 2019, 8, 170
(B)
Figure 7. Cont.
Foods 2019, 8, 170
(C)
Figure 7. Effects of EM01 on mRNA expression level of lipid metabolism-related genes in HFD-induced
obese mice. (A) Liver. (B) Epididymal adipose tissue. (C) Abdominal subcutaneous fat. Data are
presented as the mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01
and *** p < 0.001 vs. HFD-CTL. FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase-1; SREBP-1c,
sterol regulatory element binding protein-1c; PPARγ, peroxisome proliferator-activated receptor γ;
DGAT1, diglyceride acyltransferase; UCP, mitochondrial uncoupling proteins; ACOX1, peroxisomal
acyl-coenzyme A oxidase 1; PPARα, peroxisome proliferator-activated receptor α; ACS1, acetyl CoA
synthetase 1; CPT1b, carnitine palmitoyltransferase 1b.
Foods 2019, 8, 170
4. Discussion
In recent years, many side effects have been reported for drugs used for therapeutic purposes,
and numerous clinical studies have examined natural products and natural product-derived
compounds [26,27]. In this study, we confirmed the anti-obesity effect of three natural materials.
In previous studies based on these materials, N. nucifera L was found to be a medicinal plant that is
not only useful for treating gastritis, bleeding, diarrhea, hemorrhoids, and enuresis, but also contains
various biologically active components such as polyphenolics, flavonoids, and tannic acid [28]. SOD,
CAT, GST played as defence means against the reactive oxygen species in biological systems. TBARS are
formed as a byproduct of lipid peroxidation. This material was reported to have anti-oxidative activity
by increasing the levels of SOD, CAT, GST and decreasing TBARS level in liver [29] and anti-obesity
activity in HFD-induced mice or anti-obesity activity for inducing apoptosis in 3T3-L1 adipocytes,
but few studies have examined these effects [30]. Morus alba L, which is prepared for medicinal use
such as treating headache, fever, and cough, shows pharmacological activities, particularly antidiabetic
effects on lowering blood sugar [31]. Raphanus sativus is an herbaceous plant belonging to the
cruciferous family and has been reported to have excellent anti-obesity efficacy [32]. Recent studies
have demonstrated the anti-obesity activity of a single material, but the mechanism action of this
material in a mixture has not been evaluated.
We selected EM01, which contains a high content of Nelumbo nucifera [16], among the 26 mixtures
(13 kinds of hot water extracts and 13 ethanol extracts) through the antioxidant and anti-obesity
experiments at in vitro model. EM01 treatment has an anti-obesity effect at in vivo model using
C57BL/6J obese mice. The weight of HFD-induced mice was effectively lower and the weight of the
adipose tissue was significantly decreased compared with the control.
Type 2 diabetes is one of the most common disabilities caused by obesity. Obesity directly affects
insulin function, which causes glucose to enter the cell membrane for energy metabolism, resulting in
insulin resistance and type 2 diabetes [33]. A glucose tolerance test was conducted to investigate the
glucose processing ability. EM01 effectively lowered the blood glucose level. This result was similar to
those of the glucose tolerance test using neferine an alkaloid compound extracted from N. nucifera
seed [34].
EM01 reduced the serum lipid profile (TC, HDL-cholesterol, LDL-cholesterol, TG) of HFD-induced
obese mice. It may be useful for preventing dyslipidemia induced by obesity [35]. AST, ALT,
and creatinine are toxicity indicators of the liver and kidney [24]. EM01 reduced the levels of these
markers in the serum and improved the function of each organ.
Adipocytes not only play a role as energy reservoirs but also regulate endocrine function.
Adipokine, a hormone specifically expressed and secreted from adipocytes such as adiponectin and
leptin, is involved in fatty acid and glucose metabolism. IGF-1 plays an important role in regulating
adipose tissue growth. These adipokines interact closely with each other and regulate the hormonal
system in the body [36]. EM01 significantly increased the adiponectin level and reduced leptin and
IGF-1 level in serum. According to a previous study [37], glucose and lipid metabolism are regulated
by insulin signaling. Glucose and fatty acid enter cells and are metabolized for glycogenesis and
β-oxidation, respectively. Based on these results, our study confirms that EM01 improves the process
of bringing glucose and fatty acid into the cells, resulting in anti-obesity and anti-diabetic effects.
Adipogenesis is the process of cell differentiation by which pre-adipocytes become adipocytes.
PPARγ is a key factor in adipogenic transcription [1]. In this study, it was found that mRNA expression
of PPARγ in the liver tissue was reduced by administration of EM01. Also, the expression levels of FAS,
DGAT1, SCD-1, leptin, SREBP1c were decreased, while the expression levels of UCP2, PPARα, ACOX1,
and adiponectin increased in the EM01, Q3OG treatment group in the liver. These results suggest
that EM01 treatment inhibits adipogenesis of 3T3-L1 adipocytes and improves fatty acid oxidation.
EM01 regulated lipid metabolism in epididymal adipose tissue and abdominal subcutaneous fat.
Our results demonstrate that EM01 has an anti-obesity effect in 3T3-L1 adipocytes and C57BL/6J
obese mice.
Foods 2019, 8, 170
5. Conclusions
In conclusion, we observed the anti-obesity activity of EM01, which is an optimal mixed material
containing N. nucifera L., M. alba L., and R. sativus in vitro and in vivo models. EM01 reduced lipid
accumulation and weight gain, fat mass, serum lipid concentration, and mRNA expression levels of
lipid-metabolism-related genes in HFD-induced obese mice. Therefore, our findings show that EM01
has potential as an effective material for anti-obesity treatment.
Author Contributions: W.-S.S., S.-I.C., B.-Y.C., S.-H.C., X.H., H.-D.C., J.-H.C., S.-H.K., B.-Y.L., I.-J.K. and O.-H.L.
designed research; W.-S.S., S.-H.K. performed research and analyzed the data; W.-S.S., O.-H.L. wrote the paper.
All authors read and approved the final manuscript.
Funding: This research was funded Technology Development Program (C1013823-01-01) funded by the Ministry
of SMEs and Startups (MSS, Korea).
Acknowledgments: This work was supported by the Technology Development Program (C1013823-01-01) funded
by the Ministry of SMEs and Startups (MSS, Korea); NRF (National Research Foundation of Korea) Grant funded
by the Korean Government (NRF-2018-Fostering Core Leaders of the Future Basic Science Program/Global Ph.D.
Fellowship Program); The basic Science Research Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education (NRF-2017R1D1A3B06028469) and has been worked with the support
of a research grant from Kangwon National University in 2017.
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foods
Article
Use of Phycobiliproteins from Atacama Cyanobacteria
as Food Colorants in a Dairy Beverage Prototype
Alexandra Galetović 1, *, Francisca Seura 2 , Valeska Gallardo 2 , Rocío Graves 2 , Juan Cortés 1 ,
Carolina Valdivia 3 , Javier Núñez 3 , Claudia Tapia 3 , Iván Neira 3 , Sigrid Sanzana 3 and
Benito Gómez-Silva 1
1 Departamento Biomédico, Laboratorio de Bioquímica, Facultad de Ciencias de la Salud and Centre for
Biotechnology and Bioengineering (CeBiB), Universidad de Antofagasta, Avenida Angamos N◦ 601,
Antofagasta 1270300, Chile; juancortesgonzalez.96@gmail.com (J.C.); benito.gomez@uantof.cl (B.G.-S.)
2 Departamento de Ciencias de los Alimentos y Nutrición, Universidad de Antofagasta, Avenida Angamos N◦
601, Antofagasta 1270300, Chile; fran.ncisca@hotmai.com (F.S.); nutricionistavale.gd@gmail.com (V.G.);
Rociograves@hotmail.com (R.G.)
3 Departamento de Tecnología Médica, Universidad de Antofagasta/Avenida Angamos N◦ 601,
Antofagasta 1270300, Chile; carovaldiviab@gmail.com (C.V.); javier.ignacio.ng@gmail.com (J.N.);
claudiafran.t@gmail.com (C.T.); ivan.neira@uantof.cl (I.N.); sigrid.sanzana@uantof.cl (S.S.)
* Correspondence: alexandra.galetovic@uantof.cl; Tel.: +56-55-2637054
Received: 25 January 2020; Accepted: 3 February 2020; Published: 24 February 2020
Abstract: The interest of the food industry in replacing artificial dyes with natural pigments has
grown recently. Cyanobacterial phycobiliproteins (PBPs), phycoerythrin (PE) and phycocyanin
(PC), are colored water-soluble proteins that are used as natural pigments. Additionally, red PE
and blue PC have antioxidant capabilities. We have formulated a new food prototype based
on PBP-fortified skim milk. PBPs from Andean cyanobacteria were purified by ammonium
sulfate precipitation, ion-exchange chromatography, and freeze-drying. The stability of PE and
PC was evaluated by changes in their absorption spectra at various pH (1–14) and temperature
(0–80 ◦ C) values. Purified PBPs showed chemical stability under pH values of 5 to 8 and at
temperatures between 0 and 50 ◦ C. The antioxidant property of PBP was confirmed by ABTS
(2,2 -Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical ion scavenging,
and FRAP (Ferric Antioxidant Power) assays. The absence of PBP toxicity against Caenorhabditis
elegans was confirmed up to 1 mg PBP/mL. Skim milk fortified with PE obtained a higher score
after sensory tests. Thus, a functional food based on skim milk-containing cyanobacterial PBPs
can be considered an innovative beverage for the food industry. PBPs were stable at an ultra-high
temperature (138 ◦ C and 4 s). PBP stability improvements by changes at its primary structure and
the incorporation of freeze-dried PBPs into sachets should be considered as alternatives for their
future commercialization.
Keywords: cyanobacteria; phycobiliproteins; natural pigment; phycoerythrin; phycocyanin;

food colorant
1. Introduction
Cyanobacteria are a large, diverse and ancient group of ubiquitous Gram-negative prokaryotes
that are found in terrestrial or aquatic habitats. They perform oxygenic photosynthesis and colonize
freshwater, marine and brackish waters and soils and rocks from drylands. They also are found
at extreme environments that are subjected to high ultraviolet radiation, high or low temperatures,
desiccation, and nutrient deficiencies [1,2]. Cyanobacteria can be found as free-living unicellular or
filamentous microorganisms and as photobionts in symbiotic association with fungi. Members of

Foods 2020, 9, 244
some filamentous genera (e.g., Anabaena and Nostoc) have heterocysts—cells specialized in atmospheric
nitrogen fixation. Cyanobacteria are an important source of natural products with interest from
the pharmaceutical and biotechnological industries [3,4]. Particularly, cyanobacterial pigments have
attracted attention for their use in the food, textile and cosmetic industries [5,6].
Three types of cyanobacterial water-soluble phycobiliproteins (PBPs), C-allophycocyanin (APC),
phycocyanin (PC), and C-phycoerythrin (PE) are organized in phycobilisomes in the photosynthetic
apparatus. PE, PC and APC act as antennae pigments with absorption maxima at 562, 615, and 652 nm,
respectively [7]. PBPs contain linear tetrapyrrolic chromophores (bilins) that are covalently bound
to apoproteins via cysteine residues [7,8]. The ability of PBPs to act as free radical scavengers has
been demonstrated to be centered on their tetrapyrrolic systems, supporting their use in the food,
cosmetics and pharmaceutical industries, as well as their use as fluorochromes in biomedical research.
In addition, PBPs isolated from various cyanobacterial species also have beneficial effects as anticancer,
neuroprotective, anti-inflammatory, anti-allergic and hepatoprotective biomolecules [9–15].
Color in food has an important impact on consumers since it is one of the first characteristics
we perceive from a product. Additionally, colored food is attractive, and color allows for better
identification and selection among similar products. Coloration helps to relate water with food; for
example, yogurt, animal or vegetal milk (e.g., soybean, coconut, or almonds) with fruits such as
strawberries (reddish), blue berries (purple), and melon (green). Then, consumers would consider
ingesting these types of food as beneficial to their well-being even though they may not have fruits [16].
Artificial dyes are stable at different temperatures, pH and light regimes, maintaining their
coloration for long periods. Some synthetic dyes have been approved for their use in the food industry;
however, some of them have been reported to be neurotoxic, mutagenic, and genotoxic (lemon-yellow
tartrazine), damaging in the liver and the kidney (brilliant blue), and triggering biochemical changes
and cancer in the thyroid gland (cherry-red erythrosine) [17–19]. As such, the demand for natural
dyes in the food industry has grown in recent years due to the toxicity of artificial colorants [20].
There is great interest in finding non-harmful alternative pigments, especially blue pigments such
as PC, which has the capability to scavenge hydroxyl ions in order to avoid lipoperoxidation [21].
Natural dyes can be considered renewable and sustainable bioresources with minimal environmental
impact [22]. These eco-friendly, presumably mostly non-toxic, natural colorants could have applications
in other industrial sectors like the cosmetics or pharmacological industries [23–26]. Other studies have
shown that some natural colorants from plants have health-promoting properties in the human diet,
like natural carotenoids that provide beneficial biological effects such as antioxidant and anticancer
properties [27,28].
Cyanobacterial PBPs are natural pigments used as colorants in some food products; for example,
aqueous extracts of non-purified blue PC from Spirulina have been added in ice creams, yogurts, isotonic
beverages, confectionery, and jellies [29]. Particularly, bright blue PC has been selected over others
less-bright natural colorants such as gardenia blue and indigo in confectionery production [30–32]. Red
PE has been mostly used as a fluorescent probe in biomedical studies, rather than in the food industry.
Spirulina (Arthrospira platensis) has been used as a source of PBPs for additions to food products;
however, this microorganism mostly produces PC. The Andes wetlands in northern Chile are a source
of microbial communities that include PC and PE producing cyanobacteria. Isolated strains Nostoc
sp. Caquena (CAQ-15), LLA-10 and Nostoc sp. Llayta (LLC-10) from Andes wetlands, above 3000 m
of altitude, predominantly accumulate red PE, blue PC, and a purple fluorescent mixture of both,
respectively. In addition, the CAQ-15 strain modulates its PBP content by complementary chromatic
adaptation [33]. Here, we report the use of these isolated Nostocaceae strains for the purification of PBPs
and their application as colorants and antioxidant molecules in the formulation of dairy functional
prototypes. Additionally, we report results on the stability, antioxidant capabilities and toxicity of the
purified PBPs, as well as sensory tests of the final prototypes. This work represents the first step in the
use of PBPs from Andean cyanobacteria as ingredients for the food industry.
Foods 2020, 9, 244
2.1. PBPs Stability under Different Temperature Regimes

The stability of the purified PBPs from the cyanobacterial strains LLC-10 and CAQ-15 were
measured as the concentration of the remaining non-denatured PBPs after incubation at various
temperature and pH regimes. Nearly 80% of the PC and PE proteins from both strains were stable
after 72 h of incubation at temperatures from 10 to 21 ◦ C. The denaturation of the PC and PE proteins
increased to nearly 50% after 24 h of incubation at 26 to 53 ◦ C (Table 1 and Figure 1). These proteins
showed total denaturation after 48 h of incubation at temperatures over 55 ◦ C (Table 1 and Figure 1).
PC from both strains had a similar response to temperature; however, PE from the LLC-10 strain
reached 50% denaturation at 35 ◦ C, while PC required a lower temperature (26 ◦ C) to reach the same
level of denaturation. Additionally, PE was a thermally more stable protein (Table 1). Purified PE from
the LLC-10 strain and PC from the LLA-10 strain were subjected to incubations at 138 ◦ C for 4 s in order
to evaluate changes in stability after this heat treatment. Based on changes in their visible absorption
spectra, only 10% and 15% denaturation were observed at the PE and PC solutions, respectively.
The thermal stability shown by PBPs from the Atacama cyanobacterial strains was consistent
with the denaturation profiles that are expected for mesophilic proteins and also for more stable
proteins from thermophilic cyanobacteria [34]. PC denaturation from Spirulina platensis occurred after
incubations over 45 ◦ C at pH 7 [35–37]; the bleaching of PBPs from mesophilic cyanobacteria and algae
was observed at temperatures between 60 and 65 ◦ C [38]. Additionally, PC from Anabaena fertilissima
PUPPCC 410.5 was unstable at 42 ◦ C with a 50% loss after 4 days; this protein was very stable and
maintained its antioxidant properties at 4 ◦ C over 6–9 days [39,40]. Moreover, PC from Spirulina
fusiformis was denatured at temperatures above 70 ◦ C [41,42]. Though the PBPs from Phormidium
rubidium A09DM were stable at temperatures from 4 to 40 ◦ C, their corresponding absorptions at their
maximal wavelengths decreased 2–4 fold at 60 to 80 ◦ C [41]. Comparatively, PBPs from thermophilic
cyanobacteria had better temperature stability than PBPs from mesophilic cyanobacteria; for example,
PE from Leptolyngbya sp. KC 45 maintained 80% of its antioxidant activity after exposure to 60 ◦ C for
30 min [43]. Additionally, PC from Thermosynechococcus elongatus TA-1 was stable between 4 and 60
◦ C at a pH range of 4 to 9, but PC denaturation occurred at temperatures over 75 ◦ C [44]. Likewise,
the thermostable PC from the Synechococcus lividus PCC 6715 strain that was isolated from a hot spring
maintained 90% and 70% stability at 50 ◦ C for 5 h and two weeks, respectively [45]. Additionally, PC
from the thermophilic red algae Cyanidioschyzon merolae showed a midpoint denaturation at 83 ◦ C and
pH 5, with a half-life of 40 min [46].
Future work will consider the isolation of thermophilic cyanobacteria from a thermal spring
in the Atacama region and the purification of their phycobiliproteins. The biochemical properties
of these PBPs and relevant genetic studies would provide new unidentified protein resources for
biotechnological applications.
Table 1. Interpolation of temperatures to evaluate protein stability. The remaining non-denatured

phycobiliproteins content was expressed as percentage (0%, 50% or 80%) of the control condition
at 0 ◦ C. Each value shown represents the interpolated temperature at 24, 48 and 72 h of incubation.
The standard deviation for LLC-10 (PC), LLC-10 (PE), CAQ-15 (PC) and CAQ-15 (PE) were 9.3 to 13.4,
13.6 to 14.2, 15.1 to 16.4, and 15.72 to 18.6, respectively.
Percentage of Remaining Phycobiliproteins at Different Interpolated Temperatures (◦ C)

Strain PBP 0% 50% 80%
24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h
PC 84.7 54.3 52.8 42.3 27.1 26.4 16.9 10.9 10.6
LLC-10
PE 106.9 76.9 70.4 53.4 38.5 35.2 21.4 15.4 14.1
PC 68.1 57.8 49.5 34.0 28.9 24.7 13.6 11.6 9.9
CAQ-15
PE 82.7 62.5 52.5 41.4 31.3 26.2 16.5 12.5 10.5
Foods 2020, 9, 244
Figure 1. Effect of the temperature on the stability of phycocyanin (PC) and C-phycoerythrin (PE)
phycobiliproteins from the cyanobacterial Nostoc sp. Llayta (LLC-10) and Nostoc sp. Caquena (CAQ-15)
strains. The stability of the proteins was expressed as mg/mL of the remaining native phycobiliproteins
after 24 to 72 h of incubation. (*) significant, ns: no significant with respect to the reference group at 24 h.
2.2. PBPs Stability under Different pH Regimes

Based on their absorbance spectra, PE and PC from the LLC-10 strain were stable at pH 5 to 8. At
pH 5, PE and PC showed absorbance maximum at 565 and 620 nm, respectively (Figure 2). At an acidic
pH (1 to 3), a PBP precipitation was observed (Figure 2a, lower left), showing a wide non-characteristic
profile of their absorption spectrum (Figure 2a, upper left). Additionally, the incubation of PBPs at an
alkaline pH (9 to 14) rendered uncolored solutions with a change in their absorption spectra due to
protein denaturation (Figure 2b, lower right).
Both, acidic or alkaline pH values alter the electrostatic and hydrogen bond interactions among
amino acid residues in proteins; in PBP, this effect translates into structural changes in chromophores
and the apoproteins [41]. The stability of Atacama PBPs from the LLC-10 strain at pH 5–8 was similar
to PC from Spirulina platensis at pH 4–6 [35–37]. In addition, the addition of preservative molecules
such as citric acid, sugars and calcium chloride improve PBP stability [47–49]. Blue PC from Spirulina
platensis increased stability in the presence of citric acid at 35 ◦ C over 15 days [50]. Then, non-toxic PBP
stabilizers should be explored in depth to expand the use of these proteins in the food industry.
Foods 2020, 9, 244
Figure 2. Effect of pH on phycobiliprotein stability. The stability of mixed solutions of PE plus PC

from the LLC-10 strain was evaluated at an acidic (a), neutral and basic pH (b) range. Changes in
absorption spectra, coloration and solubility are presented. These experiments were run in triplicate.
The information provided in this figure corresponds to one complete experiment. The application of
the Kolmogorov–Smirnov goodness-of-fit test showed that the inequality hypothesis was significant
(p < 0.05) among the distribution functions for the absorption of each pH condition.
2.3. Antioxidants Activity

The antioxidant capabilities of purified PE and PC have been previously demonstrated.
Additionally, the antioxidant power of PC has been related to its ability to sequester hydroxyl
ions avoiding lipo-peroxidation [9,21,51–54].
The antioxidant activity of the purified Atacama PBPs was evaluated by two assays, ABTS and
FRAP, and the results are shown in Table 2. The methanol extracts from the cyanobacterial LLA-10,
CAQ-15 and LLC-10 strains showed antioxidant activity values of 195 ± 38 to 717 ± 60 μmoles Trolox
equivalent (TE)/100 g fresh mass by the ABTS assay (Table 2). Purified phycocyanin (PC-LLA-10),
phycocyanin (PC-CAQ-15), and phycoerythrin (PE-CAQ-15) showed antioxidant activities between 2
and 3 μmoles TE/100 mg pigment (Table 2). These results indicate that PBPs and the methanol
extracts from Atacama cyanobacteria have an antioxidant power comparable to fruits such as
mulberry, pineapple and passion-fruit [55]. Further support was obtained from the FRAP assay
(Table 2). Consequently, cyanobacteria from the Atacama Desert are an innovative source of functional
natural antioxidants that have a potential protective role against oxidative stress and biotechnological
applications in the food, pharmaceutical and cosmetic industries.
Table 2. Antioxidant activity of purified phycobiliproteins and methanol extracts from the Atacama
native cyanobacterial strains CAQ-15, LLC-10 and LLA-10. The antioxidant capabilities were evaluated
by the ABTS and FRAP assays and expressed as TEAC (Trolox equivalent antioxidant capacity). TE:
Trolox equivalents; PE: Phycoerythrin; and PC: Phycocyanin. The assays were conducted in triplicate,
and the results are shown as the mean values with the corresponding standard deviation.
Sample ABTS FRAP

Methanol extract μmoles TE/100 g fresh biomass
CAQ-15 717 ± 61 50.23 ± 1.64
LLC-10 641 ± 95 12.63 ± 0.73
LLA-10 195 ± 38 13.14 ± 1.52
Phycobiliprotein μmoles TE/100 mg phycobiliprotein
PE-CAQ-15 198 ± 45 0.92 ± 0.15
PC-CAQ-15 312 ± 56 1.55 ± 0.10
PC-LLA-10 205 ± 41 2.50 ± 0.15
Foods 2020, 9, 244
2.4. Toxicity of Phycobiliproteins against C. elegans

The nematode Caenorhabditis elegans offers several advantages as an emerging model in
environmental toxicology. It is easy and inexpensive to culture in the laboratory, it has a short
life cycle that allows for short-time span experiments, and there is increasing evidence on its genetic
and physiological similarity with mammals, so results related to its use have the potential to predict
possible effects in higher animals [56,57]. Our work showed that PBPs that were purified from the
LLC-10 strain (genus Nostoc) were not toxic to C. elegans; the nematode survival was 100% at all
concentrations used; comparatively, ivermectin, a nematicidal drug, showed a 100% mortality (Figure 3),
which is in agreement with the information provided by Ju et al. (2014) on other cyanobacterial
pigments [58].
Figure 3. Toxicity of phycobiliproteins against the nematode Caenorhabditis elegans. Toxicity test of
a mixed solution of PE plus PC from the LLC-10 strain was evaluated at a phycobiliprotein (PBP)
concentration from 0.125 to 1.0 mg/mL. Ivermectin (0.3 mg/mL) was used as a nematicidal control drug.
The nematode M-9 buffer was used as a control without phycobiliproteins. Quadruplicate tests were
carried out for 24 h at 18 ◦ C.
2.5. Sensory Test

Prototypes of skim milk that were fortified with the phycobiliproteins PC or PE purified from
two Atacama cyanobacterial strains were the functional foods that were tested by a volunteer team
by using an acceptability hedonic scale (Figure 4). The results of the sensory evaluation showed that
there were no statistically significant differences between the prototypes. However, the parameters’
appearance (related to the color reached by the prototype) and texture were the best valued by judges.
The appearance of the prototypes had a good acceptance (mean score 3.7) that was only surpassed by
texture. The highest score at the sensory test was obtained by the skim milk that was fortified with PE
(prototype N◦ 2).
Several reports have shown a wide acceptance for food products that are supplemented
with microalgal natural pigments, given the improvements in color and antioxidant properties,
e.g., chlorophyll and carotenoids from Chlorella vulgaris and Haematococcus pluvialis [59,60]. In addition,
microalgae have been incorporated into dairy products as a source of bioactive and coloring compounds,
with good acceptability, particularly in texture and appearance [61]. In this study, texture and
appearance stood out among all parameters tested. Therefore, a PE-fortified food would provide
health benefits to consumers.
Foods 2020, 9, 244
Figure 4. Sensory test for a PBP-containing dairy product. (a) Three skim milk prototypes were fortified
with PE and PC: Prototype N◦ 1 (PC from the LLC-10 strain at 120 mg%); Prototype N◦ 2 (PE from the
CAQ-15 strain at 13 mg%; Prototype N◦ 3 (PE from the LLC-10 strain at 140 mg%). (b) Sensory test
by a volunteer team evaluating four sensory factors (appearance, smell, taste and texture) that used
a consumer acceptability 5-point hedonic scale: 1—dislike extremely; 2—dislike slightly; 3—neither
like nor dislike; 4—like slightly; and 5—like extremely) (c) Final sensory evaluation scores for four
attributes of prototypes P1, P2 and P3.
2.6. Microbiological Assays

All prototypes were subjected to a microbiological control to evaluate the potential presence of
Enterobacteriaceae. All prototypes were free of microbial contaminant such as coliforms, Salmonella
and Shigella.
3.1. Strains and Culture Conditions of Cyanobacteria

Diazotrophic cyanobacteria Nostoc sp. Llayta (LLC-10) and Nostoc sp. Caquena (CAQ-15), were
isolated from wetlands at the XV Region in northern Chile. The strains were cultured in an Arnon liquid
medium [62] without additions of combined nitrogen at 30 ◦ C under continuous white fluorescent light
irradiation (180 μE m−2 s−1 ) and continuous aeration with air enriched with 1% (v/v) CO2 . Cultures
were harvested by centrifugation (10 min; 6000 rpm, rotor Sorvall SS-34) at the exponential phase
of growth (15 days). The liquid cultures rendered approximately 1.0 g of wet weight per 160 mL
of culture.
3.2. Phycobiliprotein Purification

PBP purification was conducted according to the method of Ranjithak and Kaushik [63] with
modifications. Briefly, cyanobacterial cell suspensions were digested overnight with lysozymes at
a final concentration of 1.0 mg/mL in a 50 mM phosphate buffer of pH 7.2 at 37 ◦ C. Next, the cell
suspensions were sonicated in a water–ice bath for 2 min at 15-s intervals (Microson Ultrasonic Cell
Disruptor XL, Farmingdale, NY, USA). One volume of the sonicated extract was diluted with one
volume of phosphate buffer and centrifuged at 10,000 rpm for 15 min (RC 5B Plus Sorvall SS-34,
Newtown, USA). The PBP-rich supernatants were recovered, and 500 μL of aliquots were used to obtain
the corresponding 280–700 nm UV/VIS absorption spectrum. The selective precipitation of proteins
from the remaining supernatants was conducted by adding ammonium sulfate, with continuous
stirring at 4 ◦ C to reach a 0%–35% saturation and a 35%–60% saturation to enrich precipitates with PE
and PC, respectively. After 24 h, each protein precipitate was resuspended in 1.0 mL of phosphate
buffer and dialyzed in 12,000 Da cut-off cellulose dialysis tubing (Sigma, Steimheim, Germany) against
distilled water at 4 ◦ C. Aliquots of the dialyzed PBP solutions were used to obtain the corresponding
280–700 nm absorption spectra. The absorption maxima 620 nm (PE), 565 nm (PC) and 650 nm (APC)
were used to compute their concentration according to the following equations:
Foods 2020, 9, 244
[PC] (mg/mL) = ([(A615 − A730) - 0.476 (A652 − A730)] × 1/5.34)
[APC] (mg/mL) = ([(A652 − A730) - 0.208 (A615 − A730)] × 1/5.09)
[PE]] (mg/mL) = ([((A562 − A730) - 0.241 [PC] − 0.849 [APC]] × 1/9.62)
PBPs were further purified by ion exchange chromatography by using a DEAE

(Diethylaminoethyl-cellulose) column (7.0 by 1.5 cm). Two or three milliliters of a PBP-dialyzed
solution was loaded in the column and eluted with a 0.03–1.0 M NaCl gradient at room temperature.
One-milliliter fractions were collected, and the colored fractions were pooled and scanned to obtain the
UV (Ultraviolet)–Vis (Visible) absorption spectra. Concentration and purity were inferred by using the
corresponding equations. The collected purified PBPs were lyophilized and stored at −20 ◦ C until used.
3.3. PBPs Stability

Triplicates of each purified PBP solution were incubated in a 50 mM phosphate buffer of pH 7.2 at
temperatures from 0 to 80 ◦ C over 72 h. Protein stability was evaluated in triplicate assays at room
temperature in a 50 mM sodium phosphate buffer adjusted to pH 1 to 14. Changes in PBP concentration
were obtained from the corresponding UV–Vis absorption spectra every 24 h. In addition, purified
PE from the LLC-10 strain and PC from the LLA-10 strain were subjected to incubations at 138 ◦ C for
4 s; visible absorption spectra were obtained before and after the heat treatment to evaluate changes
in stability.
3.4. PBPs Antioxidant Capabilities

The biomass from cyanobacterial strains Nostoc sp. Llayta (LLC-10) and Nostoc sp. Caquena
(CAQ-15) at the exponential growth phase was recovered by centrifugation (4000 rpm, 10 min; rotor
Sorvall SS-34) and washed twice with a 0.9% NaCl solution. The washed cell pellets were extracted
with 4.0 mL of 70% methanol and vortexed at maximal speed for 2 min in a Genic2 multitube holder
(Daigger Sci. Ind., model G560E, Bohemia, NY, USA). The methanol extracts were sonicated on a
water–ice bath (Microson Ultrasonic Cell Disruptor XL, Farmingdale, NY, USA) for 3 min at 10-s
intervals and clarified by centrifugation (4000 rpm, 10 min), and then the supernatants were saved.
The methanol extracts were filtered through 0.2 μm SFCA (Surfactant-Free Cellulose Acetate) syringe
filters (Ultra Cruz) and stored at −20 ◦ C. The antioxidant capacity of the methanol extracts and the
purified PBP was performed by ABTS and FRAP assays according to Re et al. (1999) and Benzie and
Strain (1996), respectively [64,65]. Results were expressed as μmoles of Trolox equivalents per 100 g of
fresh mass or milligram of pigment.
3.5. Toxicity Assays

Wild-type C. elegans var. Bristol-N2 and the Escherichia coli strain OP50 were obtained from the
Caenorhabditis Genetics Center (St. Paul, MN, USA). The nematode was plate-propagated as previously
described [66]. C. elegans var. Bristol-N2 was cultured on nematode growth agar that was seeded with
either E. coli OP50 or E. coli strain B, following the procedure reported by Brenner [67]. Gravid worms
were gently shaken at room temperature in 10 volumes of a fresh 1% NaClO/0.5 M NaOH solution.
Carcasses and other debris were dissolved after 5–10 min, and resistant eggs (50% to 100% viable eggs)
were collected and washed several times in an M-9 buffer [46]. The M-9 buffer was prepared with 1.5 g
of KH2 PO4 , 3.0 g of Na2 HPO4 , 2.5 g of NaCl, 0.5 mL of 1 M MgSO4 , and sterile distilled water to a final
volume of 500 mL [68]. Eggs were deposit in agar plates for 48 h, and young worms (larval stage 4
and 5) were harvested and resuspended in the M-9 buffer.
Foods 2020, 9, 244
3.6. Preparation and Sensory Analyses of Dairy Prototypes

Ten mL of skimmed milk were mixed with purified PBP pigments at a final concentration of
1.2 mg PC/mL and 0.3 or 1.4 mg PE/mL. The skim milk that was used in this work was a commercial
liquid product prepared by Colun (Cooperativa Agrícola y Lechera de la Unión Ltd., La Unión, Chile)
with a content of 3.3% of total protein, 0.05% total fat, 4.7% carbohydrates, 32 mg% sodium, 115 mg%
calcium and 90 mg% phosphorus. According with the manufactures, this skim milk was processed by
UHT technology (Ultra High Temperature) at 138 ◦ C for 4 s. The acceptability of PBP-containing dairy
prototypes was evaluated by sensory preferences. The study used skimmed milk as a common matrix,
and three dairy prototypes were designed: Prototype N◦ 1 (PC from the LLC-10 strain), Prototype N◦ 2
(PE from the CAQ-15 strain) and Prototype N◦ 3 (PE from the LLC-10 strain). A five-point hedonic
scale test was used to measure appearance, smell, texture and flavor. The tests involved ten university
students as impartial judges who did not have previous training in sensory-type analyses. Consumer
acceptability was measured with five-point hedonic scale (1—dislike extremely; 2—dislike slightly;
3—neither like nor dislike; 4—like slightly; 5—like extremely). Data were subjected to variance analysis
(ANOVA) with a significance level of p < 0.05.
3.7. Microbiological Analyses

Total coliforms, Shigella and Salmonella detection in the prototypes were performed in a BBLTM
MacConkey II Agar (BD, Le Point Declaix, France), a selective and differential medium for the detection
of coliforms and enteric pathogens. In addition, an XLD Agar (Xylose–Lysine–Deoxycholate Agar, BD,
Le Point Declaix, France) was used to detect Salmonella and Shigella. Samples of each prototype were
seeded on these agar plates and incubated at 37 ◦ C for 24 h [69].
3.8. Statistical Analyses

Sensory test data were analyzed with an analysis of variance (ANOVA) with α = 0.05. The PBP
temperature stability data were gathered from two independent experiments, each in triplicate, and a
non-parametric method was used in a stability test for comparisons of means values among groups
(α = 0.05). The Shapiro–Wilk normality test was done to determinate if the dataset is well-modeled
by a normal distribution. Since the data did not show a normal distribution, a Kruskal–Wallis test
was performed.
This research was approved by the Research Ethics Committee, CEIC, Universidad de Antofagasta
(Document 044/2017).
4. Conclusions
PBPs purified from two Andean cyanobacteria from northern Chile showed chemical stability at a
pH range from 5 to 8, and temperatures between 0 and 50 ◦ C. The pigmented proteins from the LLC-10
strain had no toxic effects against C. elegans. The highest score at the sensory test was obtained by skim
milk that was fortified with PE.
Colors are always attractive to children and induce consumption. The skim milk was selected to
incorporate blue PC and red PE because of its low-fat content and its contribution to human health
with vitamins, minerals and proteins, particularly to the overweight population.
We conclude that PBPs are natural proteins that can be used as colorants in the formulation
of functional food products based on skim milk in the replacement of added artificial colorants.
In addition, the native cyanobacterial PBPs have an appropriate level of antioxidant activity, and we
propose their potential use as an innovative source of pigments in the pharmaceutical, cosmetic and
food industries.
Finally, the UHT pasteurization (138 ◦ C, 4 s) of Atacama cyanobacterial PBPs induced a minor
denaturation of PE and PC; therefore, they can be added before the skim milk pasteurization process.
However, milk usually is heated over 50 ◦ C, and, in order to avoid the loss of added PBP coloration
Foods 2020, 9, 244
while maintaining bacteriological safety, we propose two alternatives for the use of cyanobacterial
PBPs at high temperatures in dairy products before pasteurization. One is the application of molecular
biology and genetic tools on PBP genes from mesophilic and thermophilic cyanobacteria in order to
increase their stability for biotechnological processes and the safety of new functional foods. The second
alternative is the use purified freeze-dried PBPs that are enclosed in sachets that can be added as
powder to already pasteurized dairy food before their consumption, avoiding protein denaturation
and the loss of antioxidant activity.
Author Contributions: Conceptualization, A.G.; methodology, A.G., I.N. and S.S.; investigation, A.G., F.S., V.G.,
R.G., C.V., J.N., C.T., I.N., J.C., S.S.; writing—original draft preparation A.G.; writing, reviewing and editing, A.G.,
I.N. and B.G.-S. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Semillero de Investigación, Universidad de Antofagasta (grant number:
SI-5305); CONICYT-Chile (grant number: CeBiB FB-0001); Financiamiento Asistente de Investigación, VRIIP,
Universidad de Antofagasta MINEDUC-UA (grant numbers: ANT1855 y ANT1856); Convenio Marco para
Universidades Estatales, Ministerio de Educación Chile (grant number: NEXER ANT 1756).
Acknowledgments: Special thanks to Milton Urrutia, Universidad de Antofagasta, for his assistance with the
statistics used in this work. We thank Catherine Lizama, Microbiology Laboratory, Universidad de Antofagasta
her support in the microbiological analyses.
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foods
Article
A Microethnographic and Ethnobotanical Approach
to Llayta Consumption among Andes
Feeding Practices
Mailing Rivera 1 , Alexandra Galetović 2 , Romina Licuime 1 and Benito Gómez-Silva 2, *
1 Departamento de Educación, Facultad de Educación, Universidad de Antofagasta,
Antofagasta 124000, Chile; mailing.rivera@uantof.cl (M.R.); rlicuime1505@gmail.com (R.L.)
2 Departamento Biomédico, Facultad Ciencias de la Salud, and Centre for Biotechnology and Bioengineering,
CeBiB, Universidad de Antofagasta; Antofagasta 1240000, Chile; alexandra.galetovic@uantof.cl
* Correspondence: benito.gomez@uantof.cl; Tel.: +56-9-98297844
Received: 29 October 2018; Accepted: 28 November 2018; Published: 9 December 2018
Abstract: Llayta is a dietary supplement that has been used by rural communities in Perú and
northern Chile since pre-Columbian days. Llayta is the biomass of colonies of a Nostoc cyanobacterium
grown in wetlands of the Andean highlands, harvested, sun-dried and sold as an ingredient for
human consumption. The biomass has a substantial content of essential amino acids (58% of total
amino acids) and polyunsaturated fatty acids (33% total fatty acids). This ancestral practice is
being lost and the causes were investigated by an ethnographic approach to register the social
representations of Llayta, to document how this Andean feeding practice is perceived and how
much the community knows about Llayta. Only 37% of the participants (mostly adults) have had
a direct experience with Llayta; other participants (mostly children) did not have any knowledge
about it. These social responses reflect anthropological and cultural tensions associated with a lack
of knowledge on Andean algae, sites where to find Llayta, where it is commercialized, how it is
cooked and on its nutritional benefits. The loss of this ancestral feeding practice, mostly in northern
Chile, is probably associated with cultural changes, migration of the rural communities, and very
limited access to the available information. We propose that Llayta consumption can be revitalized
by developing appropriate educational strategies and investigating potential new food derivatives
based on the biomass from the isolated Llayta cyanobacterium.
Keywords: Andean microalgae consumption; Atacama; cyanobacteria; Llayta; microethnography;

Nostoc
1. Introduction
The abundance and diversity of organisms in the Atacama Desert are severely limited by the
high levels of desiccation and ultraviolet light [1,2]. In the Andes Mountains highlands, biodiversity
is higher and plants have been used for centuries by local communities for feeding, foraging and
ethnomedicine [3–8]. Microalgae and cyanobacteria are part of the Andes biodiversity but seldom
acknowledged. Based on their nutritional and digestive benefits, microalgae and cyanobacteria
(i.e., Chlorella, Dunaliella, Arthrospira and Nostoc) have been part of the human diet in South America,
North America, Asia and Africa. Also, some species are natural resources for a variety of organic
molecules with high interest to the biotechnological industry (proteins, amino acids, vitamins,
polyunsaturated fatty acids, pigments) [3,4,9,10]. Edible members of the Nostoc genus are found
in China where Nostoc flagelliforme has been consumed as a delicacy for centuries but its collection is
prohibited today due to over-exploitation [11,12]. In South America, an indigenous foodstuff harvested
in the Andes wetlands, known as Llayta, is the dry biomass of macrocolonies of a cyanobacterium from

Foods 2018, 7, 202
the genus Nostoc (Figure 1). Llayta consumption is a practice that can be traced back to pre-Columbian
times and it has been recorded in documents from the 17th century [13,14] and, in a more recent
botanical report [15].
TACNA Tropic of
Capricorn
PUTRE
ARICA
IQUIQUE
ANTOFAGAS
Figure 1. Locations, in southern Peru and northern Chile, where information on Llayta was acquired.
Thus, the genus Nostoc has been an old component of the human diet in South America, and it
continues to be used today as a food additive in northern Chile (Arica y Parinacota and Tarapacá
Regions) and in southern Peru (Tacna City) (Figure 2) [5,6,8,16–18]. However, and based on preliminary
interviews, this ancient culinary legacy is disappearing and it is already unknown by the urban
communities from other areas of the region; for example, at Antofagasta, the major coastal city in
northern Chile, nearly 400 km south of Iquique (Figure 2).
Figure 2. A dry colony of Llayta obtained at a major food market in Arica, Chile. (Bar: 1 cm).
Our report provides the results of a microethnographic study conducted to learn how much
people know about Llayta and their perception on this ancestral Andean ingredient, and is meant to
Foods 2018, 7, 202
be complementary to biochemical studies done on Llayta [4]. This work was based on the following
considerations: (i) Llayta consumption is a feeding practice transmitted through generations in the
rural Andean world of South America, without reports of adverse effects on human health; (ii) Llayta
consumption is an old culinary legacy that is disappearing in regions of South America; (iii) Llayta is a
nutritional ingredient containing essential amino acids (58% of total amino acids) and polyunsaturated
fatty acids (33% total fatty acids); (iv) the prevalence of undernourishment in South America; (iv) Llayta
consumption can be revitalized with appropriate educational strategies and new food derivatives can
be developed from the biomass of the isolated cyanobacterium from Llayta [4,9,18,19].
We propose that this microethnographic approach will help us to explore explanations for an
apparent decrease in Llayta consumption, and to provide arguments and suggestions for the revitalization
of this feeding practice.
2.1. The Microethnographic Study

The microethnographic study on Llayta was designed to learn about the direct or indirect
knowledge people have about Llayta by collecting social representations [20] from interviewing
participants, including drawings prepared by children. The main expressions about Llayta were
ethnographically registered and analyzed in order to explain the social worlds built by persons about
their understanding of the surrounding natural, social and cultural environment [21].
2.2. Sample for the Microethnographic Study

Observations were carried out during the first half of 2014 in Tacna (Peru) and Putre (Chile)
(Figure 2). Tacna is a Peruvian city located at the frontier between Peru and Chile with an active
commercial exchange with Arica and Iquique, two coastal cities in Chile. Putre is a rural village at
2500 m above sea level in the Andes Range in northern Chile, close to sites where Llayta is harvested.
The participants were 19 active members of their community who were informed about the origin
and purpose of the study. Their selection was based on their willingness to participate anonymously
in the interviews.
The participants were 12 fourth-grade students (7 boys and 5 girls, 9–10 years old) and three
middle-aged adults (three males and two females) from Putre, Chile. Participants from Tacna, Peru,
were two vendors (one female and one male) working at the main food market of the city. Interviews
and observations were conducted at sites normally used by the participants (street, market place,
school, and hotel).
Oral interviews were conducted with all participants in order to learn their direct or indirect
knowledge about Llayta (origin, physical description, uses, places where Llayta grows and is sold, its
quality as food). In addition, children were also asked to draw representations of Llayta in order to
evaluate how close their depictions were from the real subject.
3. Results
3.1. The Vocable Llayta: Alternative Names and Their Meaning

Llayta is the Aymara name that refers to colonies of a cyanobacterium that grows in the Andes
highlands and is consumed by rural and urban communities in South America (Figures 1 and 2).
Alternative names for Llayta can be found in several languages: Spanish, Quechua, Kunza and
Mapudungun (Table 1). The variety of names for the vocable Llayta stresses the cultural and
anthropological diversity of representations associated with this feeding practice.
Foods 2018, 7, 202
Table 1. The Llayta vocable: alternative names, their ethnic origins and meaning.
Name Language Comments Reference

Cushuro
Llullucha
Murmuta The author did not indicate the language of origin of the words. [12]
Crespito
Yrurupa
Chuncoro “Vna yerua negra de comer, frutilla fe llama Chun-coro,o Murmunta”
Aymara [3]
Murmunta (Edible black herb called Chuncoro or murmuta)
“(Prunus capulí Cav.) Cerezo. La infusión de las hojas de esta planta se
Murmunta usa como laxante. Sus frutos en Aymara se llaman”.
Aymara [22]
Chuncuru (The leaves infusion is used as a laxative. Their fruit are named
in Aymara)
Cerezo Aymara “s. Cerezo. Plumas coloridas (10)” (Colored feathers) [23]
“Bot. Cerezo. 2. Plumas coloridas, en Bolivia. Bot. Cochayuyo de agua
dulce y del mar (p.e. alga comestible).
Quchayuyo
Aymara Vte. QUCHAYUYU, MURMUNTA” [24]
Murmunta
(Colored feathers in Bolivia. Bot. Macroalga from freshwater and sea
water) (edible alga))
“una cianobacteria comestible, se encuentra en riachuelos y lagunitas
del bofedal ubicado entre Isluga y Colchane, en las cercanías de la
frontera entre Bolivia y la Región de Tarapacá en Chile”.
Chungullu Quechua [7]
(An edible cyanobacterium, found in small rivers and lakes of wetlands
located between Isluga and Colchane, near the border between Bolivia
and the Tarapacá Region in Chile)
“ . . . , probablemente derivado de su morfología, hábitat y uso.
Murmunta, Aymara En Aymara: hierba de las ciénagas como granillos negros”.
[7]
Chuncuro Quechua ( . . . derived probably from its morphology, habitat and use.
In Aymara: black grain herb from wetlands)
Luche “ . . . , es un símil de una alga roja, marina y comestible”.
Mapuche [7]
(lucha) (..., is like a marine edible red alga)
“De llullu para referirse a formas vegetales que empiezan a
Yullucha Aymara desarrollarse o se pasman”. [8]
(From llullu, to refer to plants starting or have stopped their growth).
“Nombre atacameño de la cianobacteria comestible Nostoc.
Tchuckula Planta acuática que hay en la cordillera”.
Kunza [8]
Chucula (Name given by the Atacameños people to the edible cyanobacterium
Nostoc. Aquatic plant found in the Andes Range).
Aymara “Cianobacterias del género Nostoc”
Yoyo [9]
Quechua (cianobacteria from the genus Nostoc).
“Cianobacteria acuática de bofedales, procedentes de Chela. Se usa
para la comida (caldo con papas chuño).
Chungulle, Se indicó que “hay uno que se come, es especial. Se lava y se seca”.
Quechua [9]
chungullo (Aquatic cyanobacterium from Chela wetland. It is used as food, in
soups with potatoes. It was mentioned that there is an edible one which
is special. It is washed and dried).
“Símil de un alga roja comestible para designar a
un alga verde-azulada de agua dulce, también comestible (Nostoc)”.
Luche Mapuche [9]
(Similar to an edible red alga, it is (Used to indicate an edible,
freshwater blue-green alga, (Nostoc)).
“Nombre de la cianobacteria Nostoc en Ollagüe, posiblemente
aludiendo a su carácter comestible. Planta acuática comestible”.
Yoyo Aymara [9]
(Name given in Ollagüe to the cyanobacterium Nostoc,
possibly due to its edibility).
3.2. How Much People Know about Llayta

All participants were interviewed to assess the type and level of knowledge they have on Llayta.
Table 2 provides extracts of the answers given by 10 participants (7 adults and 3 students). Only 8 of
the 19 participants had some perception about Llayta; the others (58%) lack any knowledge about it.
Foods 2018, 7, 202
The extracts in Table 2 corroborate that 7 participants have had personal experience of Llayta, i.e., direct
knowledge. Only one teacher expressed indirect knowledge about Llayta since the information was
second-hand (Table 2). Compared with adult participants, the oral expression of knowledge used by
the fourth-grade students from Putre to refer to Llayta were few or absent. When asked to draw an
image of Llayta, 11 out of 12 students were willing to participate and their drawings were far from a
correct depiction of the colonies. As an exception, one student emphasized that his mother used to
cook Llayta and her drawing was the closest image to it. Another student said: “no, yo no” (No, I do
not (know Llayta)). A third student asked: “¿Esa es la Llayta? (Is this Llayta?), referring to a drawing
made by another student. Table 2 is a compilation of the representations of Llayta.
Table 2. Transcripts of interviews conducted to adult and students participants about their knowledge
on Llayta.
Informant Type of Knowledge about Llayta

DIRECT KNOWLEDGE:
“Ahí huapé súcuros; de Súcuro se trae; de súcuro de ahí al fondo pues; ahí arriba de Puno. Otros
Saleswoman at the
caballeros traen y ahí compramos; para picante. Si, picante prepara rico ahí comen”.
food market, Tacna,
(There, huapé súcuros; it is brought from Sucuro; down there; there, above Puno. Other people bring it
Peru.
and we buy it to make “picante”—A local dish; yes, a tasty picante is prepared up there).
“Esta es nacional; ésta la traen de Camaná. Esta es de río muestra -y muestra Llayta-, es más rica y esa es
Salesman at the
de mar -muestra cochayuyo; a tres soles. Esto jefe chángalo, muélelo, jugo”.
food market, Tacna,
(This is Peruvian; it is brought from Camaná. This from a river, it is better and this one is from the sea; it is
Peru.
worth three Peruvian new sols. Cut it and grind it for juice, boss).
“Para el consumo lo comemos eh; la Llayta es un tipo de alga de mar, de agua dulce y de mar también hay.
Lo traen y lo hacen secar, y seco lo venden en los negocios; para cocinarlo se la remoja. Los peruanos los
comen la Llayta y el cochayuyo. De la altura, de Puno, de Juliaca, bofedales eso está en la altura, en agua
Professional cook, dulce en los ríos crece por ahí, bueno acá en la frontera con Perú, tripartito, Visviri, ahí también crece”.
Chile. (We eat it for consumption, eh!; Llayta is a kind of marine alga; it is from freshwater and also marine.
They bring it, dry it and sell it dried; they soak it before cooking. Peruvians eat Llayta and cochayuyo.
From the highlands, from Puno, from Juliaca, from wetlands, which are in the highlands, in freshwater
rivers, it grows around; right here, at the three parties’ border, it also grows at Visviri).
“Llayta, arriba hay, arriba; Parinacota, ahí si hay Llayta; Llayta come..., el segundo come bonito, así que
coce para . . . , es como carne para . . . , se prepara eso como carne, como picante cocino acá. Ahí no se pa’
Middle age woman, qué sea, en Parinacota hay río de esa, ahí florece”.
Putre, Chile. (Llayta is from up there; Llayta is at Parinacota; Llayta is eaten . . . , nice as a second dish . . . , it is cooked
. . . , it is like meat . . . , it is prepared as meat . . . , I cook it here as picante. I do not know what it is used
for over there; in Parinacota there is a river where it grows).
“La Llayta es de por acá también. La Llayta se usa para el picante. La he comido no más, pero no sé qué
me ha hecho; acá hacen mucho picante de pata con guata y Llayta. Hay en las lagunas, en Caqueña, en
Director, school at Tacna y aquí arriba Caquena”.
Putre, Chile. (Llayta is from here too. Llayta is used in picante. I have only eaten it; I do not know the effects on me;
people right here prepares a lot of picante with meat and Llayta. There are some ponds, in Casqueña, in
Tacna and up here in Caquena).
“¡no! y los picantes de guatita, ahí le ponen la Llayta . . . es un musgo que se trae de Caquena; es un
Teacher 1, at school musgo parecido al cochayuyo, es la misma que venden en el agro”.
in Putre, Chile. (No! Llayta is added at the picante dishes . . . , it is a moss brought from Caquena; it is a moss similar to
cochayuyo marine macroalga, it is the same one that is sold at the food market).
Student 1, at school “Ah! yo sí, porque mi mamá cocina. En Caquena”.
in Putre, Chile. (Mm! I do, since my mother cooks it. At Caquena . . . ).
INDIRECT KNOWLEDGE:
“Eh . . . Yo no las he visto pero sí me han dicho que ahí en la laguna está la Llayta” pero de verla no.
Teacher 2 at school En Caqueña en Tacna y aquí arriba Caquena”.
in Putre, Chile. (Eh . . . , I have not seen it, but I have been told that Llayta is at the pond, but I have not seen it.
At Caquena, in Tacna and up here in Caquena).
WITHOUT KNOWLEDGE:
Student 2, at school
“No, yo no”. (No, I do not . . . know Llayta).
in Putre, Chile.
Student 3, at school
“¿esa es la Llayta?” (Is that Llayta?)
in Putre, Chile.
Foods 2018, 7, 202
3.3. Fields of Representations for Llayta

Table 2 shows the extracts from the ethnographic registries. These are the descriptions and
references that sustain the field of representation of Llayta for 7 adult participants, which can be
organized in the following 3 semantic fields:
3.3.1. What is it?

“Es un musgo que se trae de Caquena”; “es un musgo parecido al cochayuyo, es la misma que
venden en el agro”. “Ésta es nacional”; “la Llayta es un tipo de alga de mar, de agua dulce y de mar
también hay”.
“It is a moss brought from Caquena”; “it is a moss similar to cochayuyo (seaweed), it is the same
that is sold at the market”, “This is national”; “Llayta is a kind of marine alga; from freshwater and
also from the sea”.
3.3.2. Where is it from?

“De súcuro se trae”, “De súcuro de ahí al fondo pues”; “Ahí arriba de Puno”; “ésta es de río”;
“en Parinacota hay río de esa, ahí florece”; “la Llayta es de por acá también”; “Hay en las lagunas,
en Caqueña en Tacna y aquí arriba Caquena”; “de la altura, de Puno, de Juliaca, bofedales eso está en
la altura, en agua dulce en los ríos crece por ahí, bueno acá en la frontera con Perú, tripartito, Visviri,
ahí también crece”; “Llayta, arriba hay, arriba”; “Parinacota, ahí si hay Llayta”; “la Llayta es de por acá
también”; “en Caquena”; “Eh . . . Yo no las he visto pero sí me han dicho que ahí en la laguna está la
Llayta, pero de verla no”; “en Caquena”.
“It is brought from Sucuro”; “from Sucuro, back there”; “from Puno, up there”; “this is from a
river”; “at Parinacota, there is a river where it blooms”; “Llayta is from here too”; “it is from ponds, at
Caquena, in Tacna, and up here in Caquena”; “from highlands, at Puno, Juliaca, wetlands, this is at
the highlands, it grows in freshwater rivers, well, here at the border with Peru, Visviri, where it also
grows; “Llayta, it is up there, up high”; “Llayta is at Parinacota, for sure”; “Llayta is from here too”;
“at Caquena”; “Eh . . . , I have not seen it but I was told that over there at the pond, there is Llayta, but I
have not seen it”; “at Caquena”.
3.3.3. What is it for?

“Para picante”; “si picante prepara rico ahí comen”; “los peruanos los comen la Llayta y el
cochayuyo”; “Llayta come... el segundo come bonito, así que coce para . . . es como carne para . . . se
prepara eso”; “como carne, como picante cocino acá”; “la Llayta se usa para el picante”; “la he comido
no más, pero no sé qué me ha hecho”; “¡no! y los picantes de guatita”; “ahí le ponen la Llayta . . .
”; “lo traen y lo hacen secar, y seco lo venden en los negocios para cocinarlo se la remoja”; “ah yo sí,
porque mi mamá cocina”.
“For making picante dish”; “yes, picante dish is good, they eat it”; “Peruvian people eat Llayta
and cochayuyo”; “Llayta is for consumption . . . , so you cook it . . . , it is like meat . . . ; like meat, like
picante I cook it here”; “Llayta is used for picante”; “I have eaten it but I do not know how to prepare
it”; “it is brought here, it is dried and it is sold dry to restaurants”; “it is soaked before cooking”; “yes I
know it, my mother cooks it”.
When asked for places where Llayta can be found, one informant from Tacna, Peru, used the
vocable “chuncuru” (an Aymara synonym for Llayta) instead of using “Sucuru”, the right geographic
site to where Llayta can be found. This mistake can be explained by the phonetic similarity between
both words.
Foods 2018, 7, 202
4. Discussion
4.1. Microethnographic Aspects

The ethnographic goal of this study was to document the anthropological and cultural tensions
found in the social representations of the Llayta feeding practice and relate them to the knowledge
and value given by the communities to Andean algae.
Ethnographic registries allow the collection of evidence and social representations from people
on a particular subject, to discover what people think, believe, and know about their surroundings,
and understand how people see it and fit it in their particular interpretations of realities [21,25,26].
The knowledge people may have on a particular natural situation is a good example of where social
representations can be collected and interpreted from social and cultural perspectives [26]. Also,
descriptions and references from participants are essential in the field of representation for an event of
ethnographic interest [22].
In the study of the Llayta feeding practice, the ethnographic registry can be supported by
anthropological, socio-cultural, and nutritional referents [20]. The first two provide information on the
meaning(s) of the term Llayta, the identification of sites where Llayta grows naturally, and where it is
commercialized and consumed.
Our results indicated that nearly 40% of the participants declared they knew Llayta and described
it as a moss or an alga, without clarifying whether it grows in fresh water or seawater (Table 2).
They also provide names for the sites of origin of Llayta: Parinacota, Putre, Caquena and Visviri in
Chile, and Sucuro, Juliaca and Puno in Peru (Table 2, Figure 2). The participants knew that Llayta is
used to prepare “picante”, a typical dish from rural areas in the Andes; however, they were unaware
of its nutritional properties. All fourth-grade students from Putre did not know Llayta (11 out of
12 students; 58% of the participants of this study).
It is of considerable concern to confirm that a large proportion of the participants, all young
people, did not know Llayta. This apparent loss may be explained by considering the impact of
new technologies on rural life, cultural changes and migration from rural regions into urban centers,
as described for the Aymara people in northern Chile [24,27].
The cultural and anthropological tensions observed in this study stemmed from a lack of knowledge
on the following subjects: (a) conceptual limitations to explain what is an Andean alga or a microalga;
(b) geographic locations where Llayta can be found and commercialized; (c) weak descriptions to
explain how Llayta is cooked; and (d) the benefits obtained from its consumption. Also, this low or
absence of knowledge on Llayta can be explained by how people perceive and relate to the world
around them and, most stressfully, by a probable extinction of an Andean cultural practice. The massive
ignorance about Llayta found in discussions with children in Putre is a clear example of it.
These anthropological tensions are indication of a paucity of information on Llayta. There
is therefore an urgent need to educate people about all the cultural and nutritional knowledge
accumulated on Llayta, so that it can be properly valued as a nutritional foodstuff.
4.2. Biochemical Characterization of Llayta

Llayta consumption is a traditional practice whose future revitalization can be supported by
ethnographic information and evidence on the nutritional quality of Llayta [4].
The first biochemical evaluation of colonies of Nostoc cells from Peruvian wetlands were published
by Aldave-Pajares [16,17]. Later, Gómez-Silva et al. [18] registered the proximal composition of Llayta
colonies sold and consumed in Chilean territory, and also for the biomass from the isolated Llayta
cyanobacterium. Both studies are in agreement on the total protein (30–35% w/w) and carbohydrate
(50–60% w/w) content of the Andean Nostoc biomasses. More recently, it was informed that 60% of
Llayta amino acids can be classified as indispensable; total lipids accounted for 2% of the biomass
dry weight; and 32% of total fatty acids were polyunsaturated fatty acids, Vitamin E content was
4.3 mg% w/w, total polyphenols was 64 mg (as equivalent to gallic acid), with an antioxidant
Foods 2018, 7, 202
activity of 17.4 μmoles (as equivalent to Trolox), and total fiber content was 56% of dry weight [4].
Galetovic et al. [4] inferred that Llayta biomass is a nutritious dietary ingredient.
One reason for the need for safety assessments of foods and food ingredients based on microalgae
and cyanobacteria biomasses to protect public health is the potential presence of cyanobacterial toxins
active at low doses (e.g., microcystin and nodularin). In particular, Arthrospira platensis (Spirulina)
is considered a safe food based on centuries of human consumption [23]. Comparatively, some
species of the Nostoc genus have toxic members that synthesize microcystine-like cyanotoxin [28].
However, the genome of the colony-forming Nostoc strain isolated from the Llayta biomass did not
show the presence of mycE, a key gene in the microcystine biosynthetic pathway, rendering it as a
non-toxic Nostoc strain [4,28]. In addition, the absence of epidemiological records associated with Llayta
consumption diminishes but does not remove the potential presence of toxic secondary metabolites in
this cyanobacterial biomass [4].
5. Conclusions
Llayta consumption is a feeding practice with a know-how that has been transmitted through
generations in the rural Andean world of South America, without untoward effects on human health.
Nevertheless, this ancestral feeding legacy is being lost in young generations from northern Chile.
New educational and anthropological strategies must be developed if there is interest in promoting
the preservation and value of this traditional feeding practice and cultural legacy.
Llayta is a nutritious dietary ingredient for human consumption. This is supported by the
absence of adverse epidemiological evidence, but also with interdisciplinary studies that complement
ethnographic records with biochemical information.
Caution on the amount of Llayta consumed daily, based on its arsenic content, must be stressed.
However, mass growth of the cyanobacterium isolated from Llayta under controlled conditions would
provide arsenic-free biomass for the formulation of new food products. This biotechnological approach
would revitalize the use of and add value to this ancestral food ingredient for the benefit of not only
Andean communities, but also the whole population of South America and the world.
Author Contributions: Conceptualization, B.G.-S. and M.R.; Data curation, M.R. and R.L. Funding acquisition,
B.G.-S. and A.G.; Investigation, A.G.; Methodology, M.R.; Supervision, B.G.-S. and A.G.; Validation, R.L.;
Writing—original draft, B.G.-S. and M.R.; Writing—review and editing, B.G.-S.
Funding: This research was funded by Universidad de Antofagasta, Chile, grant number SI-5305, and
CONICYT-CHILE, grant number CeBiB FB-0001.
Acknowledgments: The authors would like to thank Celedonio Maron Chura, Biblioteca de Antropología Andina
(Instituto para el Estudio de la Cultura y Tecnología Andina, Arica, Chile) for his useful comments.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
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Development of Food Chemistry Natural Products and Nutrition Research

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Development of Food Chemistry Natural Products and Nutrition Research

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Development of

Special Issue Editors

ISBN 978-3-03936-461-9 (Hbk)

About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Antonello Santini and Nicola Cicero

Francesco Giuseppe Galluzzo, Gaetano Cammilleri, Alessandro Ulrici, Rosalba Calvini,

Helena Maria Pinheiro-Sant’Ana, Pamella Cristine Anunciação, Clarice Silva e Souza,

Archimede Rotondo, Giovanna Loredana La Torre, Giacomo Dugo, Nicola Cicero,

Inga Matulyte, Aiste Jekabsone, Lina Jankauskaite, Paulina Zavistanaviciute,

Joanna Zielińska-Wasielica, Anna Olejnik, Katarzyna Kowalska, Mariola Olkowicz and

Mailing Rivera, Alexandra Galetović, Romina Licuime and Benito Gómez-Silva

Received: 3 April 2020; Accepted: 4 April 2020; Published: 12 April 2020

Keywords: foods; food analysis; nutrition; natural products; food supplements;

Foods 2020, 9, 482; doi:10.3390/foods9040482 www.mdpi.com/journal/foods

Received: 4 November 2019; Accepted: 5 December 2019; Published: 12 December 2019

Foods 2019, 8, 676; doi:10.3390/foods8120676 www.mdpi.com/journal/foods

2. Materials and Methods

2.1. Reagents and Standards

2.2. Sample Collection and Preparation

2.4. Validation of the GC-FID Method

2.5. Data Collection and Statistical Analysis

3.1. Fatty Acid Proﬁles

T. pisana T. pisana C. aspersum C. aspersum E. vermiculata E. vermiculata

3.2. Fatty Acids of Raw Samples

3.3. Fatty Acids after Heat Treatment

3.4. Multivariate Analysis

Received: 3 April 2019; Accepted: 14 May 2019; Published: 16 May 2019

Keywords: Fortunella margarita; citrus; carotenes; xanthophylls; β-citraurin-laurate; β-cryptoxanthin-

Foods 2019, 8, 166; doi:10.3390/foods8050166 www.mdpi.com/journal/foods

2. Materials and Methods

2.2. Collection and Preparation of the Samples

2.3. Moisture Analysis

2.4. Extraction of Carotenoids

2.5. Analysis of Carotenoids by HPLC-DAD-APCI-MS

2.6. Identiﬁcation and Quantiﬁcation of Carotenoids

3. Results and Discussion

3.1. Carotenoids Qualitative Proﬁle of Brazilian Kumquat

Compound Identiﬁcation Rt (min) PDA (λnm) MS (APCI-) m/z

Figure 4. Chemical structures of β-cryptoxanthin-laurate and β-citraurin-laurate, main carotenoids

3.2. Carotenoids Quantitative Proﬁle of Brazilian Kumquat

Table 2. Carotenoid content in kumquat from Brazil (peel + pulp).

Content in Lyophilized Content in Fresh Carotenoid

Author Contributions: Conceptualization, H.M.P.-S., P.C.A.; Methodology, C.S.e.S., G.X.d.P.F.; Writing-review

Received: 12 November 2019; Accepted: 5 December 2019; Published: 9 December 2019

Foods 2019, 8, 659; doi:10.3390/foods8120659 www.mdpi.com/journal/foods

2. Materials and Methods

2.2. Plant Materials

2.3. Isolation of Steam-Distilled Essential Oil

2.4. Isolation of Free Volatiles and Glycosidically Bound Volatiles

2.5. Gas Chromatography (GC) and GC–Mass Spectrometry (GC–MS) Analysis

2.6. Determination of Total Phenolic Content

2.7. Antioxidant Activity

2.7.1. Preparation of Sample

2.7.2. DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Free Radical-Scavenging Activity

DPPH radical-scavenging activity (%) = (1 − A0 /A1 ) × 100

2.7.3. ABTS (2,2 -Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid)) Free Radical-Scavenging Activity

ABTS radical scavenging activity (%) = (1 − A0 /A1 ) × 100

2.7.4. Ferric-Reducing Antioxidant Power (FRAP)

2.8. Statistical Analysis

3. Results and Discussion

3.1. Chemical Composition of the Steam-Distilled Essential Oil (SDEO) Fraction

Concentration (μg/100 g dw) 3)

Concentration (μg/100 g dw) 3)